Tài liệu Báo cáo khoa học: Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3 doc

14 620 0
Tài liệu Báo cáo khoa học: Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3 doc

Đang tải... (xem toàn văn)

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Thông tin tài liệu

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]. 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 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 in vitro DNA 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-functional 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 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 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 subjecting 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 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- 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 SuperdexÒ 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 PhenomenexÒ 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. 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 2 , reached a peak in the presence of Mg 2+ 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 Mg 2+ at concentrations between 5 and 10 mm (Fig. S1). With Mn 2+ 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 Mn 2+ . Furthermore, Zn 2+ 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 65 °C 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 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 1 516 prim-pol M1 23 4 5kDa 66 29 45 36 Rep245 29 24 20 kDa 66 M12 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-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 (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 [ 32 P]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 90 °C. 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 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 0 5678910 pH 80 100 120 Mg (2+) 80 100 20 40 60 Residual activity (%) 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 AC BD 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- tion of smaller products was only observed at 80 and 90 °C, 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 + – + – +– + + ++– – 1234 16 nt 20 nt 28 nt 28 nt 20 nt 35 n t ATP ATP Temperature [°C] Rep516 C 5 50 70 65 80 Rep245 5 50 70 65 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 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 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 G U A dC dG dT dAAB CD 28-mer 0312 4 0 312 4 0312 4 0 312 4 28-mer dC dG dT dA C GU A 20-mer 20-mer Fig. 5. Rep245 has a 3¢-terminal nucleotidyl-transferase activity. TdT activity was assayed at 60 °Con5¢-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. Analysis of the pIT3 prim–pol domain S. Prato et al. 4396 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS All 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]. Mg 2+ 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 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, 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, 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 4397 we 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 vitro primase 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 [ 32 P]dATP[aP] (3000 CiÆmmol )1 ), [ 32 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- 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 =80A ˚ ) 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 )1 ÆA ˚ )1 prior to MD simula- tion. After this, it was subjected to 90 ps restrained MD (5 kcalÆmol )1 ÆA ˚ )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 [...]... 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 incubating the protein at 60, 70 and 80 °C for 30 min and then recording the CD spectra of the incubated samples at indicated temperatures Thermophilicity and thermostability Thermophilicity was evaluated in the temperature range 40–90 °C by measuring the polymerase... Milan, Italy) and cloned in pGEMTeasy vector (Promega, Milan, Italy) The nucleotide sequences of both DNA strands of the inserts were verified The NdeI–XhoI fragments were cloned into the same sites of the expression vector pET-30c(+) to obtain the recombinant plasmids pETRep245 and pETRep516, containing both an in-frame fusion with the six histidine C-terminal tag Analysis of the pIT3 prim–pol domain proteins... Bartolucci S (2006) pIT3, a cryptic plasmid isolated from the hyperthermophilic crenarchaeon Sulfolobus solfataricus IT3 Plasmid 56, 35–45 19 Delarue M, Pock V, Tordo N, Moras D & Argos P (1990) An attempt to unify the structure of polymerase Protein Eng 3, 461–467 Analysis of the pIT3 prim–pol domain 20 Braithwaite DK & Ito J (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases... 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... primer synthesis was checked by incubating an assay mix (10 lL) containing polymerase buffer, 100 lm each of CTP, GTP and UTP, and 10 lm [32P]ATP[aP], 0.25 lg of M13 mp18 ssDNA, 1.7 lm Rep245 or 0.8 lm Rep516 at 60 °C for 30 min Primer elongation was carried out at 60 °C upon further 30 min incubation in the presence of 0.2 mm dNTPs and 0.5 U of Taq DNA polymerase (Promega) To analyze the effect of temperature... 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 FEBS Journal 275 (2008) 4389–4402 ª 2008 The Authors Journal compilation ª 2008 FEBS 4399 Analysis of the pIT3 prim–pol domain S Prato et al Determination of pH and divalent ion optima for polymerase activity The influence of pH on Rep245... biochemical characterisation of the p41–p46 complex from Pyrococcus furiosus J Biol Chem 276, 45484–45490 11 Iyer LM, Koonin EV, Leipe DD & Aravind L (2005) Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm -domain proteins: structural insights and new members Nucleic Acids Res 33, 3875–3896 12 Lipps G, Rother S, Hartl C & Krauss GA (2003) A novel type of replicative enzyme... replication in the Archaea Microbiol Mol Biol Rev 4, 876–887 7 De Falco M, Fusco A, De Felice M, Rossi M & Pisani FM (2004) The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3¢-terminal nucleotidyl-transferase activity Nucleic Acids Res 32, 5223–5230 8 Lao-Sirieix SH & Bell SD (2004) The heterodimeric primase of the hyperthermophilic archaeon Sulfolobus. .. conformational energies of organic and biological molecules? J Omput Chem 21, 1049–1074 43 Ryckaert J-P, Ciccotti G & Berendsen HJC (1977) Numerical integration of the Cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes J Computat Phys 23, 327–341 44 Koradi R, Billeter M & Wuthrich K (1996) MOLMOL: ¨ a program for display and analysis of macromolecular structures... information The following supporting information is available: Fig S1 The influence of (A) divalent cations, (B) pH and (C) temperature on Rep245 polymerase activity This supporting information can be found in the online version of this article Please note: Blackwell Publishing are not responsible for the content or functionality of any supporting information supplied by the authors Any queries (other than . Molecular modeling and functional characterization of the monomeric primase–polymerase domain from the Sulfolobus solfataricus plasmid pIT3 Santina. 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

Ngày đăng: 18/02/2014, 18:20

Từ khóa liên quan

Tài liệu cùng người dùng

  • Đang cập nhật ...

Tài liệu liên quan