Furthermore, a longer variant Rep516 comprising the 1–516 N-terminal residues of the pIT3 full-length replication protein was designed and its nucleic acid synthetic activity was compare
Trang 1Molecular modeling and functional characterization
of the monomeric primase–polymerase domain from
the Sulfolobus solfataricus plasmid pIT3
Santina Prato1, Rosa Maria Vitale2, Patrizia Contursi1, Georg Lipps3, Michele Saviano4, Mose´ Rossi1,5and Simonetta Bartolucci1
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 polymerprim-ase for RNA primer de novo
synthesizing and elongation respectively [1,2]
Based on the biochemical data accumulated to date,
archaeal DNA replication involves a smaller number
of polypeptides at each stage of the process and is thus
just a simpler form of the much more complex eukary-otic replication machinery [3–6] Nonetheless, Archaea are not simply ‘mini Eukarya’ A better definition would be ‘a mosaic of eukaryal and bacterial systems with specific archaeal features’ Aspects worth men-tioning in this respect are the promiscuous nature of the nucleic acid functions performed by archaeal primases and the dual, template-dependent and
Keywords
DNA replication; pIT3 plasmid; primase–
polymerase domain; Sulfolobus; terminal
transferase
Correspondence
S Bartolucci, Dipartimento di Biologia
Strutturale e Funzionale, Universita` degli
Studi di Napoli Federico II, Complesso
Universitario di Monte S Angelo, Via
Cinthia, 80126, Naples, Italy
Fax: +39 0816 79053
Tel: +39 0816 79052
E-mail: bartoluc@unina.it
(Received 4 April 2008, revised 23 June
2008, accepted 4 July 2008)
doi:10.1111/j.1742-4658.2008.06585.x
A tri-functional monomeric primase–polymerase domain encoded by the 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 novoprimer 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.
Trang 2-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
in vitroDNA strands up to several kb in length [8–10]
Despite their unique multifunctional nature, archaeal
DNA primases share a number of features with
eukar-yal ones and are consequently subsumed within the
superfamily of structurally related proteins called
archaeo-eukaryotic replicative primases (AEPs) [11]
Primase–polymerases (prim–pols) are a novel family of
AEPs which are sporadically found in both
bacterio-phages and crenarchaeal and Gram-positive bacterial
plasmids In a recent description, they are said to be
typified by the RepA-like protein ORF904 encoded by
the pRN1 plasmid from the hyperthermophilic
archa-eon Sulfolobus islandicus [12,13] Prim–pols catalyze
both a DNA polymerase and a primase reaction
(hence the name) They are often fused with
superfam-ily III helicases or encoded by genes in proximity to
those encoding such helicases [12] It has been
sug-gested that both these primases and the associated
heli-cases are the constituent elements of the replication
initiation complex of the corresponding plasmids [12]
Available structural data on the small primase subunit
of the euryarchaeote Pyrococcus furiosus (Pfu) [14], the
S solfataricus (Sso) [15] and Pyrococcus horikoshii
(Pho) [16] heterodimeric primase complexes and the
prim–pol domain from S islandicus plasmid pRN1
[13] reveal that the novel fold in the N-terminal
mod-ules of the catalytic cores of AEPs and prim–pols is
unrelated to that of other known polymerases, whereas
the RRM-like fold encompassed by their C-terminal
units is also reported for the catalytic modules of other
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-funcfunc-tional recombinant prim–pol domain encoded by a crenar-chaeal plasmid identified to date Using an approach combining homology modeling, molecular simulations and biochemical analysis, we identified a number of structural features which are likely to account for diverse nucleic acid synthesis functions associated with the 1–245 N-terminal domain of the putative replica-tion protein from the S solfataricus plasmid pIT3 Furthermore, a longer variant (Rep516) comprising the 1–516 N-terminal residues of the pIT3 full-length replication protein was designed and its nucleic acid synthetic activity was compared with that exhibited by Rep245
Results
Homology modeling and structure–sequence analysis
The N-terminal domain comprising residues 31–245 of the orf915-encoded putative replication protein of the plasmid pIT3 was predicted to be the minimum-length sequence containing all the functionally relevant struc-tural motifs [18] This domain (without the 30 N-termi-nal residues) was modeled onto the crystallographic structure of the orf904-encoded bifunctional prim–pol domain of the archaeal plasmid pRN1 (PDB entry 1RN1) [13], which following PSI-BLAST sequence search against PDB and FUGUE server fold recogni-tion was found to be the best possible structural tem-plate In point of fact, this template was found to be the only prim–pol domain from archaeal plasmids that had been structurally characterized to date
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
Trang 3the 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
protein, i.e all regions except those with gaps⁄
inser-tions In the Rep245 model, all secondary structure template elements were conserved except the b11 strand which connects the a5 and a6 helices in the pRN1 prim–pol protein Because of a two-residue gap
in the corresponding region of the Rep245 sequence, this finding had not been predicted in phd and prof secondary structure prediction programs (data not shown) Fold stability was assessed by energy-minimiz-ing the model thus selected and subjectenergy-minimiz-ing it to 1.5 ns molecular dynamics (MD) simulation in water Snap-shots saved every 15 ps were seen to be best fitted at the heavy atom backbone level with an rmsd value of 1.04 A˚ The larger fluctuations we expected actually occurred in the 183–201 loop region, whereas second-ary structure content and distribution were found to undergo no change during the simulation Compara-tive analysis of the resulting model (Fig 1B) and the template structure revealed that two structural ele-ments which are highly conserved in prim–pol domains were absent from the prim–pol domain of pIT3: the
A
B
Fig 1 Structure-based sequence alignment
of Rep245 prim–pol domain (31–245).
(A) Sequence alignment between 1RNI and
Rep245 prim–pol domains Secondary
struc-ture elements of the Rep245 model are
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
representation 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.
Trang 4Zn-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-aged to model the entire domain without it
Another significant finding concerns the nature of
the acid residues within the active site of Rep245 The
carboxylate triad of Rep245 including the D101, D103
and D166 motif is similar to the triads of X family
DNA polymerases and terminal deoxynucleotidyl
transferases (TdTs) [21], but differs from that of the
pRN1 prim–pol which contains the D111, E113 and
D171 motif The presence of an aspartic residue in
place of the glutamic one is likely to have functional
implications: a drastic decrease in enzymatic activity
has been observed upon the mutation of aspartate to
glutamate in human terminal TdT enzyme [22]
Structure–function analysis conducted on the
Rep245 prim–pol domain also pointed to K135 and
R186 residues being potentially critical for a putative
primase activity of this domain, because these
posi-tively charged residues: (a) are not conserved in the
pRN1 prim–pol, whose domain performs no primase
activity; and (b) after the best possible fit of the Ca
atoms of the catalytic triad, are positional homologs
of the R148 and K300 residues of P furiosus archaeal
primase, both of which are known to play a pivotal
role in the activity of this protein [14] The side-chains
of the first pair of residues, i.e K135 and R148,
matched almost exactly; those of the second pair were
in close proximity The R148 residue of the
Pfu-prim-ase is part of a motif which is highly conserved in
archaeal and eukaryotic primases and is also found in
the Rep245 sequence (146-SGRGYH-151 in Pfu-prim
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 amino acids 1–245 and then including the residues pre-dicted to be responsible for the DNA polymerase and primase activities, accordingly to the homology model-ing data (Fig 2A) As described in the Experimental Procedures, the deletion gene was amplified using the PCR of the S solfataricus plasmid pIT3 [18] and then cloned into pET-30c(+) In E coli, the recombinant protein (from now on Rep245) was highly overexpres-sed as a fusion with the C-terminal six-residue histidine tail (LEHHHHHH) The Rep245 obtained from heated protein extracts was purified to homogeneity in
a two-stage process using, in succession, affinity chro-matography on HisTrap HP and anionic exchange on the Q Resource column SDS⁄ PAGE analysis revealed
a single band with an expected molecular mass of
29 kDa (Fig 2B; lane 5) To assess the quaternary structure of purified Rep245, we conducted analytical gel filtration on Superdex 75 PC 3.2 ⁄ 30 The protein was eluted at a volume consistent with a monomeric form (data not shown) As a further purification step a Phenomenex C4 (with a linear gradient 5–70% aceto-nitrile and trifluoroacetic acid 0.05%) reverse-phase column was used
In addition, a longer variant comprising the N-ter-minal residues 1–516 (Rep516) and lacking the C-ter-minal ATP⁄ GTP-binding site motif A was also designed and the truncated protein was purified under the same conditions as described for the Rep245 (Fig 2A,C; lane 1)
Biochemical characterization of Rep245 DNA 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
Trang 5The 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 Mg2+, Mn2+ and Zn2+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 MgCl2, reached a peak in the presence of Mg2+ ions
at 5 mm and was seen to diminish at higher ion con-centrations, we concluded that the activating metal preferably used by Rep245 for its DNA polymerase activity was Mg2+ at concentrations between 5 and 10 mm (Fig S1) With Mn2+ as a cofactor, the DNA polymerase activity of Rep245 was found to be optimal at lower ion concentrations (1–2.5 mm) and to decrease noticeably at increasing amounts of Mn2+ Furthermore, Zn2+ cations do not support the DNA polymerization activity of Rep245
The thermophilicity of Rep245 was characterized by investigating its polymerase activity at increasing tem-peratures utilizing the TP heteropolymeric DNA sub-strate As shown in Fig 3C, the peak reached at 65C was followed by rapid decreases in activity at higher temperatures This behavior may be traced to melting synthesis products and⁄ or enzyme inactivation A gel profile of the products is shown in Fig S1
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 80C, 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
prim-pol
M 1 2 3 4 5
kDa
66
29
45
36
Rep245 29
24
20
kDa
66
M 1 2
Rep516
Rep245 29
45
36
24
20
14.2
Fig 2 Schematic representation and production of truncated
vari-ants of the replication protein (RepA) of the plasmid pIT3 from
Sulf-olobus solfataricus, strain IT3 (A) RepA, Rep245 and Rep516
indicate the full-length residues, 1–245 and 1–516 truncated
proteins, respectively The constructs represent the C-terminally
His-tagged proteins The prim–pol domain and putative
heli-case ⁄ NTPase domain are indicated in gray and black respectively.
(B) Purification of the recombinant Rep245 protein SDS ⁄ PAGE of
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-His6-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
Trang 6Eventually, heat resistance tests conducted by
assay-ing residual polymerase activity after 15 min
incuba-tion at temperatures between 50 and 80C showed
that Rep245 was fairly stable even after incubation at
80C, when its residual activity was found to be 60%
of the corresponding level of non-preincubated samples
(Fig 3D)
Rep245 can synthesize RNA and DNA primers
Next, we addressed the question if Rep245 could display
primase activity Significantly, following incubation
with M13 mp18 single-stranded DNA in the presence of
a ribonucleotide mixture containing [32P]ATP[aP],
Rep245 was actually found to be capable of synthesizing
an alkali-labile 16-base RNA primer as well as a less
abundant 20-mer oligoribonucleotide RNA primer
for-mation was found to be a specific activity because it
was not detected in the absence of Rep245 (Fig 4A)
Surprisingly, Rep516, the longer variant comprising
the N-terminal residues 1–516 (Fig 2A,C, lane 1), was
found to be capable of de novo synthesis of larger
molecular size RNA products (Fig 4B, lane 1) These
RNA primers formed on the M13 mp18 can be
elongated by Rep516 and Taq DNA polymerase when
further incubation in the presence of dNTPs was performed (Fig 4B, lanes 3 and 4) When Rep516 was omitted, neither a ribonucleotide primer nor elongation products were observed (Fig 4B, lane 2)
Another point we set out to investigate was whether Rep245 could use dNTPs as a substrate for primer synthesis For this purpose, primase reactions with dNTPs as substrates were performed on M13 mp18 single-stranded DNA at temperatures between 5 and
90C Under these reaction conditions, the Rep245 protein was found to efficiently synthesize and elongate 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 30C; in the temperature range between 40 and 65C, 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
Temperature (°C)
0
40 50 60 70 80 90
60 80 100
Relative activity (%) 20
40
0
5 6 7 8 9 10
pH
80 100
120
Mg (2+)
80 100
20 40 60
Mn (2+)
Zn (2+)
Relative acitivity (%) 20 40 60
0
NP 50 60 70 80 Pre-incubation T (°C) ion concentration (m M )
0
0 1 2.5 5 10 50
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.
Trang 7specific activity value (0.607 nmol dNTPsÆmin)1Æmg)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-tion of smaller products was only observed at 80 and
90C, suggesting that Rep516 was more active than Rep245 in performing DNA synthesis Hence the dif-ferent efficiency in de novo RNA⁄ DNA synthesis can
be ascribed to additional residues responsible for the lesser frequency with which this enzyme is dissociated from DNA during catalysis
Taken together, these findings indicate that besides performing RNA primer synthesis activity, the Rep245 and Rep516 proteins can both incorporate dNTPs for
de novo primer synthesis and elongate these primers into larger DNA products, though the efficiency to make long products of Rep516 is higher than that of the smaller Rep245 variant and is comparable with the wild-type protein In conclusion, the Rep245 domain contains the catalytic residues required for both primase and polymerase activities
Rep245 performs 3¢-terminal nucleotidyl transferase activity
During our primase activity test, we observed that following incubation with poly(dT), Rep245 syn-thesized greater than template-length DNA primers (data not shown) To establish whether the protein could also perform a non-template synthesis function
we resolved to verify whether different 5¢-end labeled oligonucleotides underwent elongation in the presence
of unlabeled (d)NTPs For this purpose, individual DNA substrates were incubated with Rep245 and separately supplied with each of the four (d)NTPs As shown in Fig 5, Rep245 was found to preferentially 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
+
–
+ – +
–
+ +
+
+
–
16 nt
20 nt
28 nt
28 nt
20 nt
35 nt
Temperature [°C]
Rep516
C 5 50 70
Rep245
5 50 65 70 80 C
16 nt
20 nt
28 nt
dATP d
Fig 4 Primase activity of Rep245 and Rep516 proteins (A) RNA
primer synthesis Reaction mixtures, containing M13
single-stranded circular DNA, NTPs including [ 32 P]ATP[aP], and Rep245
(or Rep516), were incubated at 60 C for 30 min ss20-mer,
ss28-mer and ss35-mer oligonucleotides were 5¢ labeled with
[ 32 P]ATP[cP] and used as markers (B) Rep516 synthesized and
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 m M 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 [32P]dATP[aP] as substrates The approximate size of the
bands (in nucleotides) is indicated on the right-hand side of each
panel.
Trang 8when 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
60C, 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 been produced in a template-directed fashion More-over, the evidence that nucleotide addition was not governed by the sequence of the substrates used for these assays was further supported by the finding that Rep245, when incubated with each of the above DNA oligonucleotides, proved able to incorporate all of the four (d)NTPs tested
Discussion
In this study, we describe the structure–function analysis of a 1–245 N-terminal domain of the puta-tive replication protein encoded by the pIT3 plasmid from S solfataricus, the shortest fully functional prim–pol domain from a crenarchaeal plasmid identi-fied and characterized to date To model the N-ter-minal domain of the pIT3 replication protein encompassing residues 31–245 (i.e Rep245) we used
as a template the resolved crystal structure of the prim–pol domain of the protein ORF904 from the pRN1 plasmid of S islandicus, which had been iden-tified via both fold recognition and sequence search against the PDB data bank [13] In structural terms, the pIT3 prim–pol domain mainly differs from that
of pRN1 because it has no Zn-stem motif and lacks two disulfide bonds (one of which is located at the bottom of the Zn-stem) However, a MD simulation
on the Rep245 model showed that the absence of the two disulfide bridges did not affect the overall protein fold The Zn-binding motif is a structural feature conserved in all archaeal primase–eukaryotic primases characterized to date [13,23] By virtue of 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
A dC
dA
28-mer
28-mer
dC
20-mer
20-mer
Fig 5 Rep245 has a 3¢-terminal nucleotidyl-transferase activity.
TdT activity was assayed at 60 C on 5¢-end-labeled oligo(dT)28 (A,
B) and a random 20-mer (C, D) oligonucleotides (see Table 1 for
details of the sequence), as described in Experimental procedures.
Reaction products were separated on 20% polyacrylamide ⁄ urea
gels and radioactivity was detected by autoradiography Lanes 1–4
of each gel were loaded with reaction mixtures containing only the
indicated (d)NTPs in addition to the DNA template and the protein,
whereas lane 0 contains a control reaction without protein.
Trang 9All known DNA polymerases require divalent
cations for catalysis The main function of the metal
activator is to coordinate incoming nucleoside
triphos-phate substrates with the catalytic site of the DNA
polymerase molecule [17] Mg2+ is thought to be the
divalent metal cation employed by most polymerases
for in vivo catalysis [1] Similarly, the DNA polymerase
activity of Rep245 was found to be dependent on
diva-lent cations, especially Mg2+ 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 Mn2+ ions
at low concentrations (1.0–2.5 mm) and strongly
inhib-ited at higher concentrations The ability of
polymeras-es to use Mn2+ instead of Mg2+ as a required
cofactor is well established [25] However, the
bio-chemical properties of polymerases are altered as a
result of replacing Mg2+ with Mn2+, which reduces
substrate selection stringency and incorporation fidelity
[26]
Thermal activity analysis of Rep245 revealed an
optimal temperature of 65C, i.e 10 C lower than
the growth temperature of the natural host S
solfatari-cus strain IT3 harboring the pIT3 plasmid Hence,
additional extrinsic factors such as post-translational
modifications, compatible solutes, molecular
chaper-ones and other heat shock factors present in the S
sol-fataricus cytosol may be involved in protecting the
enzyme against thermal denaturation and guaranteeing
its performance in vivo [27] Our data clearly show that
DNA polymerase activity of the Rep245 was resistant
to heat treatment Hence, it is highly unlikely that such
a temperature-stable activity stems from an E
coli-derived protein present in the enzyme preparation
Moreover, we carried out a Rep245 mock purification
of an E coli culture expressing an unrelated protein
and were not able to detect any DNA polymerase or
primase activities
Bacterial and eukaryotic primases synthesize primers
of defined lengths regardless of template sequence
[1,2] The typical length of RNA primers produced by
the eukaryotic heterodimeric primase is 6–15
nucleo-tides [1,28] It has previously been reported that the
N-terminal (255 residues) prim–pol domain of the
pro-tein ORF904 from the archaeal pRN1 plasmid does
not retain any primase activity, although in this
bifunctional domain the same active site is responsible
for both DNA polymerase and primase activity [13]
By contrast, our study reveals that Rep245 retains its
primase activity, synthesizes primers of 16
nucleo-tides and is able to incorporate dNTPs for primer
synthesis The typical length of Rep245-synthesized
DNA primer is 16–20-mer, plus a few 28-mers DNA
products of defined lengths suggest that Rep245 is 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 supported by experimental evidence pointing to enhanced Rep245 primase activity and better synthesis product accumulation at higher temperatures In light
of these observations, we designed a longer variant comprising the 1–516 N-terminal residues (Rep516) and investigated its biochemical properties As we anticipated, in RNA⁄ DNA synthesis Rep516 proved more active than Rep245, in that it generated new and extended DNA and RNA products which were up to several kb in length
Hence we suggest that: (a) the additional 271 N-ter-minal amino acids were necessary to stabilize the grip
of the polymerase on its DNA substrate, and the enzyme is also able to perform continuous strand synthesis; or (b) the polymerase activity of Rep245 is stimulated to a large extent by inclusion of the extra-portion of the protein in Rep516
The Rep245 protein typifies the shortest functional domain among those endowed with primase and poly-merase activities
Based on the design of the Rep245 and Rep516 mutants and comparison of their polymerase activities,
Trang 10we were able to account for the promiscuous nature of
the synthesis functions performed by the prim–pol
domain and to discriminate between the functions of
in vitroprimase and polymerase
Another finding of our biochemical analysis was that
Rep245 is able to elongate the 3¢-end of DNA
mole-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-terize their multifunction nature, thus demonstrating
that the main activities required for DNA replication
are included in a single-chain polypeptide This
inde-pendent protein organization suggests a mechanistic
coupling of earlier DNA replication steps such as
primer synthesis and its elongation and, hence, the
autonomy of the plasmid from the host replication
apparatus This is particularly important for
environ-mental plasmid survival and transfer into new hosts
The promiscuous nature of the prim–pol domains might
be an atavistic feature evidencing a continuous link
between primase and polymerase activities and the
ori-ginal core replicon of primordial cells In light of this
suggestion, it seems plausible that prim–pol proteins are
evolutionary precursors acting both as primases and
DNA polymerases, whereas the proteins descended
from them evolved distinct and specific activities
Within this scenario, the structural and functional simi-larities between AEP superfamily proteins might be indicators of this evolutionary interconnection
Experimental procedures
Materials
PCR grade (d)NTPs were from Roche Applied Science (Monza, Italy) Radioactive nucleotides [32P]dATP[aP] (3000 CiÆmmol)1), [32P]ATP[aP] (3000 CiÆmmol)1) and [32P]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-ment, modeller v 6.2 [39] was used to construct 50 homology models (Q31–Q245) and their quality was assessed by using procheck v 3.5.4 [40] and the 3D profile
of insightii (Accelrys Software Inc., San Diego, CA, USA) The best model was completed by addition of all hydrogen atoms and underwent energy minimization followed by MD simulation in explicit solvent with the sander module of the amber 8 package [41], using PARM99 force field [42]
To perform MD simulation in solvent, the minimized model was confined in a truncated octahedron box (x, y,
z= 80 A˚) filled with TIP3P water molecules and counteri-ons (Na+) to neutralize the system The solvated molecule was then energy minimized through 1000 steps with the solute atoms restrained to their starting positions using a force constant of 10 kcalÆmol)1ÆA˚)1 prior to MD simula-tion After this, it was subjected to 90 ps restrained MD (5 kcalÆmol)1ÆA˚)1) at constant volume, gradually heating to
300 K, followed by 60 ps restrained MD (5 kcalÆmol)1ÆA˚)1)
at constant pressure to adjust the system density The