Two DNA polymerase sliding clamps from the thermophilic archaeon Sulfolobus solfataricus1

Article No. jmbi.1999.2939 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 291, 47±57

Two DNA Polymerase Sliding Clamps from the Thermophilic Archaeon Sulfolobus solfataricus Mariarita De Felice1, Christoph W. Sensen2, Robert L. Charlebois3, MoseÁ Rossi1,4 and Francesca M. Pisani1* 1

Istituto di Biochimica delle Proteine ed Enzimologia (C.N.R.), Via G. Marconi 10. 80125, Napoli, Italy 2

Institute for Marine Biosciences (N.R.C.), Halifax NS, Canada, B3H 3Z1 3

Canadian Institute for Advanced Research, University of Ottawa, ON, Canada 4

Dipartimento di Chimica Organica e Biologica UniversitaÁ di Napoli ``Federico II'', Via Mezzocannone, 16. 80134 Napoli, Italy

Herein, we report the identi®cation and characterization of two DNA polymerase processivity factors from the thermoacidophilic archaeon Sulfolobus solfataricus. They, referred to as 039p (244 amino acid residues, 27 kDa) and 048p (249 amino acid residues, 27 kDa), present signi®cant primary structure similarity to eukaryotic proliferating cell nuclear antigen (PCNA). We demonstrate that both 039p and 048p form oligomers in solution and are able to substantially activate the synthetic activity of the single-subunit family B DNA polymerase from S. solfataricus (Sso DNA pol B1) on poly(dA)-oligo(dT) as a primertemplate. This stimulatory effect is the result of enhanced DNA polymerase processivity, as indicated by the analysis of the elongation products on polyacrylamide gels. Activation of Sso DNA pol B1 synthetic activity was also observed on linear primed single-stranded M13 mp18 DNA as a template. By immunoblot analysis using speci®c rabbit antisera, 039p and 048p were both detected in the logarithmic and stationary phases of S. solfataricus growth curve. This is the ®rst report of the identi®cation and biochemical characterization of two distinct DNA polymerase processivity factors from the same organism. The signi®cance of these ®ndings for the understanding of the DNA replication process in Archaea is discussed. # 1999 Academic Press

*Corresponding author

Keywords: DNA polymerase; sliding clamp; thermostability; Archaea; DNA replication

Introduction DNA replication is a complex and highly coordinated process which requires in all living organisms a multimolecular apparatus (Baker & Bell, 1998). Evidence from biochemistry and genomics suggests that the molecular mechanisms of this process are evolutionarily conserved (Kornberg & Baker, 1992). Thus, our understanding of the DNA replication process would be greatly enhanced by a broad comparative analysis based on species of different phylogenetic origin. Our current knowledge of DNA replication in Archaea is very poor (Edgell & Doolittle, Abbreviations used: DNA pol, DNA polymerase; Sso, Sulfolobus solfataricus; ORF, open reading frame; PCNA, proliferating cell nuclear antigen; IPTG, isopropyl-b-Dthiogalactopyranoside; A, absorbance. E-mail address of the corresponding author: [email protected] 0022-2836/99/310047±11 $30.00/0

1997), although these organisms were proposed more than two decades ago to be the third domain of life by Woese & Fox (1977). Indeed, in past years the interest of most researchers in this ®eld was focused on the isolation and characterization of DNA polymerase (DNA pol) activities from archaeal hyperthermophilic species, mainly because of their biotechnological relevance (Perler et al., 1996). These studies revealed that the DNA polymerases of Archaea: (i) belong to the family B, which also includes eukaryotic DNA pols a, d and e; (ii) possess an associated 30 -50 exonuclease (or proof-reading) activity; (iii) display, in most cases, low processivity, that is the ability of synthesizing long stretches of DNA without dissociating from the template. The processivity is a critical enzymological parameter in order to understand the physiological role of a DNA polymerase. Indeed, DNA polymerases that in the cell normally operate on short gaps of the primer/template, as in the DNA repair reactions, are generally non# 1999 Academic Press

48

Archaeal Sliding Clamps

processive. In contrast, DNA polymerases involved in genome duplication possess high processivity. This property is conferred upon them by speci®c auxiliary factors, variously referred to as tracking proteins or sliding clamps, due to their ability to act as mobile tethers for the replicase machinery (Kuriyan & O'Donnell, 1993). The most extensively investigated sliding clamps are the b subunit of Escherichia coli DNA pol III (McHenry, 1988; Kelman & O'Donnell, 1995a), the gene 45 protein of T4 phage (Young et al., 1996) and the proliferating cell nuclear antigen (PCNA) of yeast (Burgers & Yoder, 1993) and man (Prelich et al., 1987). Eukaryotic PCNA, initially discovered as a cell cycle-dependent antigen, is the sliding clamp for the DNA polymerases d and e (for a review, see Kelman, 1997). These processivity factors, despite the low level of sequence similarity and the different oligomeric state (the E. coli b subunit is a dimer, whereas T4 gp45 and PCNA are trimers), share a very similar toroidal structure Ê , big enough to with a central hole of about 35 A encircle a duplex DNA molecule, as revealed by crystallographic studies (Kong et al., 1992; Krishna et al., 1994; Gulbis et al., 1996). Although ORFs encoding putative homologs of eukaryotic PCNA have been identi®ed in all the genome sequences of Archaea (Edgell & Doolittle, 1997), no archaebacterial processivity factors have been characterized so far. Therefore, we decided to search an ORF coding for a PCNA homolog in the almost complete genome sequence of the thermoacidophilic archaeon Sulfolobus solfataricus (Charlebois et al., 1996; Sensen et al., 1998), since a family B DNA polymerase from this organism (Sso DNA pol B1) was extensively characterized in our laboratory (Pisani & Rossi, 1994; Pisani et al., 1992, 1996, 1998). Quite surprisingly, we have identi®ed two homologs of eukaryotic PCNA in S. solfataricus genome sequence and herein we demonstrate that both these proteins: (i) possess an oligomeric structure; (ii) produce a noticeable activation of the synthetic activity of Sso DNA pol B1 by enhancing its processivity; (iii) are present in the exponential and stationary phases of the Sulfolobus growth curve. These ®ndings may contribute to dissect the DNA replication apparatus of Archaea and to thoroughly investigate its phylogenetic relationships.

Results Identification of putative PCNA-like factors in Archaea The aim of this study was to identify a PCNAlike factor that could act as a sliding clamp for the B1-type DNA polymerase from S. solfataricus. The genome of this bacterial species has been almost completely sequenced by the so-called ``Sulfolobus group'' in Halifax, Canada (Charlebois et al., 1996; Sensen et al., 1998). Survey of the S. solfataricus genome database with the program BLASTP pinpointed two ORFs (c41-039 and c41-048) potentially coding for proteins (referred to as 039p and 048p) with a certain primary structure identity to human PCNA (22 and 15 %, respectively). The size of these proteins (039p: 244 aa, 27 kDa; 048p: 249 aa, 27 kDa) as well as their theoretical isoelectric point (5.31 and 4.82, respectively) are common to most eukaryotic processivity factors (Kelman & O'Donnell, 1995b). To better investigate the signi®cance of these similarities, we produced a multiple sequence alignment of some eukaryotic PCNA factors and the putative homologs from Sulfolobus and from the other archaeal species, whose genome sequence is available, including Methanococcus jannaschii (Bult et al., 1996), Archeoglobus fulgidus (Klenk et al., 1997), Methanobacterium thermoautotrophicum (Smith et al., 1998) and Pyrococcus horikoshii (Kawarabayasi et al., 1998; see Figure 1). As shown in Table 1 the percentages of identity among the eukaryotic PCNA factors range from 70 % (for Homo sapiens and Drosophila melanogaster) to 35 % (for H. sapiens and Saccharomyces cerevisiae); whereas the sequence identities found within the group of archaebacterial proteins appear to be lower (values ranging from 42 % to 19 %). Quite unexpectedly, the primary structure identity between S. solfataricus 039p and 048p is only 19 %. Nevertheless, the similarities among the various sequences are considerable, as shown in Figure 1. In particular, the motif L/I-A-P-K/R located in the C-terminal region seems to be universally conserved. By mutagenesis studies (Fukuda et al., 1995; Mossi et al., 1997), these amino acid residues were demonstrated to be critical for the functional interaction of human PCNA with the replication factor C, the heteropentameric complex responsible for the ATP-dependent loading of PCNA onto

Table 1. Percentage of sequence identity among eukaryotic and archaeal PCNA factors Hsa Dme Spo Sce Mja Pho Mth Afu 039 048

70 50 35 25 24 29 22 22 15 Hsa

51 36 24 22 25 22 20 14 Dme

44 29 25 27 25 19 16 Spo

29 26 23 21 21 17 Sce

42 35 30 24 20 Mja

30 25 29 21 Pho

25 25 20 Mth

20 19 Afu

19 039

048

Archaeal Sliding Clamps

49

Figure 1. Alignment of PCNA sequences from eukaryotic and archaebacterial species. The computer program ClustalX (version 1.64b) was used. The organisms and the protein accession numbers are: Hsa, H. sapiens (P12004); Dme, D. melanogaster (P17917); Spo, Schizosaccharomyces pombe (Q03392); Sce, S. cerevisiae (P15873); Mja, M. jannaschii (Q57697); Pho, P. horikoshii (B71112); Mth, M. thermoautotrophicum (O27367); Afu, A. fulgidus (G69291); Sso, S. solfataricus: 039p (AJ243289); 048p (AJ243426). Amino acid residues that are identical (red) and similar (green) in all sequences or similar (blue) in 580 % of sequences are shown. Similar amino acids are grouped as LIMV, AG, YWF, DEQN, KRH and ST. The position of the b-sheets and a-helices of eukaryotic PCNA conserved fold is shown by arrows and cylinders, respectively; yellow and blue color indicates domain 1 and 2 of eukaryotic PCNA monomer, respectively (Krishna et al., 1994; Gulbis et al., 1996).

50

Archaeal Sliding Clamps

DNA (for a review, see Mossi & HuÈbscher, 1998). Another eye-catching feature of the multiple alignment of Figure 1 is that the archaeal sequences present a conspicuous gap corresponding to the loop between bD2 and bE2 of eukaryotic PCNA (Krishna et al., 1994; Gulbis et al., 1996). It has been very recently demonstrated that this loop of human PCNA is important for the interaction with DNA polymerase e (Maga et al., 1999). This ®nding is consistent with the fact that no homolog of eukaryotic DNA polymerase e has been found in Archaea so far (Edgell & Doolittle, 1997). Biochemical features of Sso DNA pol PCNA-like factors To determine whether the ORFs c41-039 and c41-048 of S. solfataricus truly encoded DNA polymerase processivity factors, the corresponding proteins 039p and 048p were produced in recombinant form and biochemically characterized. They were both found to be soluble and were puri®ed by heat precipitation steps and ionic exchange chromatography. Analysis by SDS-PAGE of the homogeneous proteins revealed a single band of about 35 kDa for 039p and 30 kDa for 048p, although their molecular mass was predicted to be 27 kDa (see Figure 2). This discrepancy could be likely due to an anomalous behavior during denaturing gel electrophoresis, as previously reported also for eukaryotic PCNA factors (Arroyo et al., 1996). The identity of the puri®ed recombinant proteins was probed by NH2-terminal sequence analysis after electroblotting from a denaturing gel

Figure 3. Estimation of 039p and 048p molecular mass by gel ®ltration chromatography. Calibration curve of the gel ®ltration column: 25 mg of puri®ed 039p ( & ) or 048p (~) were subjected to gel ®ltration chromatography, as described in Materials and Methods. Molecular mass standards (*) used are: tyroglobulin (670 kDa, not shown); 1, immunoglobulin G (158 kDa); 2, ovalbumin (44 kDa); 3, mioglobin (43 kDa); 4, vitamin B-12 (1.35 kDa).

to a PVDF membrane. The partial NH2-terminal sequences were, as expected: M-K-V-V-Y-D-D-V-RV, for 039p; and M-F-K-I-V-Y-P-N-A-K, for 048p. The native molecular mass of 039p and 048p was calculated by analytical gel ®ltration to be 110(10) kDa and 100(10) kDa, respectively (see Figure 3). Although on the basis of these results we cannot rule out that 039p and 048p form tetramers in solution, we favor the hypothesis that they have a trimeric structure by homology with eukaryotic PCNA. The observed hydrodynamic properties might likely depend on the putative ring-like, not globular molecular shape of these proteins. In fact, sedimentation analysis in preformed glycerol gradients, carried out under various pH and salt conditions, gave sedimentation coef®cient values lower than expected (3 S for 039p and 2.5 for 048p), consistent with the hypothesis that the proteins analyzed are not globular (data not shown). Effect of 039p and 048p on Sso DNA pol B1 synthetic activity

Figure 2. Puri®cation of recombinant S. solfataricus PCNA-like factors 039p and 048p. Protein samples from each puri®cation step were subjected to denaturing electrophoresis on a 10 % polyacrylamide gel. Staining was carried out using Coomassie. Lanes 1 and 4 contain crude extract (10 ml, 150 mg of protein) from IPTGinduced E. coli BL21 (DE3) cells harboring the plasmid pT7-039p and pT7-048p, respectively. Lanes 2 and 5 contain the above crude extracts (10 ml, 70 mg of protein) after the heat-treatment steps at 55 and 60  C. Lanes 3 and 6 contain a sample of 039p and 048p (10 mg) after Mono Q chromatography, respectively. The molecular mass of markers run in lane M is noted in kDa at the right.

The ability of 039p and 048p to stimulate the synthetic activity of Sulfolobus DNA polymerase B1 was tested by means of an in vitro assay with poly(dA)-oligo(dT) as a primer-template. As reported in Figure 4, both 039p and 048p are able to stimulate DNA synthesis by this enzyme in a dose-dependent manner. However, the level of maximal activation of Sso DNA pol was over tenfold by 048p and about sevenfold by 039p. To determine whether this effect was the result of enhanced processivity, the length of the reaction products was examined on polyacrylamide gels. Indeed, under the assay conditions utilized (short

51

Archaeal Sliding Clamps

Figure 4. Stimulation of Sso DNA pol B1 activity by 039p and 048p. Sulfolobus DNA polymerase was assayed using poly(dA)/(50 -32P)-end-labeled (dT) as a templateprimer and increasing amounts of 039p and 048, as described in Materials and Methods. DNA synthesis was quanti®ed by phosphorimagery. Data reported are mean values of at least two independent experiments. DNA synthesis by Sso DNA pol alone was taken as 100 %.

incubation times and high primer-template to polymerase molar ratio) primers were elongated by the DNA polymerase as the result of single binding events, so that their length was a measure of the enzyme processivity. As shown in Figure 5, under the above reaction conditions, Sso DNA pol B1 alone displays a very low, almost not detectable synthetic activity; on the other hand, in the presence of 039p (or 048p) the polymerase processivity is noticeably enhanced, so that only full-length products are observed, and this accounts for the observed increase in overall synthesis. The effect of 039p and 048p on Sso DNA pol B1 activity was tested also on primed single-stranded M13 mp18 DNA that had been linearized by using an appropriate restriction enzyme, as schematized in Figure 6(a). A noticeable stimulation of Sso DNA pol B1 synthetic function by 039p or 048p was detected also on this more natural primer-template and this effect is clearly dependent on the amount of protein factor added (see Figure 6(b) and and (c)). It can be noticed that the level of maximal activation effected by 048p is appreciably higher than that obtained in the presence of 039p, as also observed when poly(dA)-oligo(dT) was used as a primer-template.

Thermal stability of 039p and 048p The heat-resistance of 039p and 048p sliding clamp activity was monitored at 60 and at 80  C. The ability of 039p (and 048p) to stimulate Sso DNA pol B1 synthetic activity was substantially unaffected by incubation of each protein at 60  C for at least one hour; a reduction of about 40 % with respect to the initial value was observed after incubation at 80  C for 60 minutes for both proteins, as shown in Figure 7. The thermal stability features of the stimulatory effect on Sso DNA pol B1 by our 039p and 048p preparations allowed us to rule out that this phenomenon could be the result of some E. coli contaminant protein.

Detection of 039p and 048p in S. solfataricus cells

Figure 5. Enhancement of Sso DNA pol B1 processivity by 039p and 048p. The effect of 039p (or 048p) on polymerase processivity was tested on poly(dA)/ (50 -32P)-end-labeled (dT) as described in Materials and Methods. The reaction products were analyzed on denaturing polyacrylamide gel. Lane 1, template-primer alone; lane 2, 048p (0.5 mg); lane 3, 039p (0.5 mg); lane 4, Sso DNA pol B1 (4 ng); lanes 5 and 6, Sso DNA pol B1 (4 ng) and 048p (0.5 mg) or 039p (0.5 mg), respectively; lane 7, Sso DNA pol B1 (2 ng); lanes 8 and 9, Sso DNA pol B1 (2 ng) and 048p (0.5 mg) or 039p (0.5 mg), respectively.

Polyclonal antibodies directed against recombinant 039p and 048p were raised in rabbits for use in probing S. solfataricus cell extracts by Western blotting. As shown in Figure 8, this analysis reveals that the two proteins are present during both the exponential and stationary phases of S. solfataricus growth curve at roughly similar levels. In addition, the immunoblot analysis indicates that 039p and 048p are not immunologically related, since antibodies directed against one protein do not crossreact with the other. This ®nding is consistent with the low level of sequence identity between the two proteins, as previously mentioned.

52

Archaeal Sliding Clamps

Figure 7. Thermal stability of 039p and 048p. The heat-resistance of 039p and 048p sliding clamp activity was tested at 60 and 80  C. At each indicated time, samples were transferred into ice and tested for residual activity in standard conditions, as described in Materials and Methods. Data reported are mean values of two independent experiments. The elongation products synthesized by Sso DNA pol B1 were resolved in sequencing gels and analyzed by phosphorimagery.

Discussion The recent publication of the genome sequence of some archaeal species has revealed that the molecular machineries of DNA replication, gene transcription and RNA splicing of Archaea are more similar to the eukaryotic counterparts, despite the obvious ``prokaryotic character'' of the archaeal cell in morphology, chromosomal organization and most metabolic pathways. These ®ndings are consistent with Woese's proposal that bacteria separated ®rst from the common lineage that then gave rise to both Archaea and Eukarya (Olsen & Woese, 1997). Despite this knowledge, our current understanding of the DNA replication process in Archaea is very limited. In this context, an important and novel ®nding of our biochemical study is that the thermoacidophilic archaeon S. solfataricus has two PCNA-like processivity factors, that are both able to activate the B1-type DNA polymerase of this organism by greatly enhancing its processivity. Quite interestingly, the two PCNA-like factors of S. solfataricus were detected by Western blot analysis in both the exponential and stationary phases of

Figure 6. Stimulation of Sso DNA pol B1 activity on linearized single-stranded M13 DNA. (a) Diagram showing the preparation of the linear substrate used for the enzymatic assays. (b) Autoradiography of synthesis products run on 1.5 % alkaline agarose gel. Reaction mixtures contained Sso DNA pol B1 alone (lane 1), Sso

DNA pol B1 plus 0.014, 0.028, 0.07, 0.14, 0.7, 1.4 and 2.1 mg of 039p (lanes 2-8) or 048p (lanes 9-15). 32P-endlabeled EcoRI-linearized M13 mp18 double-stranded DNA was used as full length product size marker. (c) Incorporation of dNTP, quanti®ed by acid precipitation, was plotted against amounts (mg) of 039p and 048p as indicated. Data reported are mean values of at

Archaeal Sliding Clamps

Figure 8. Immunoblot analysis. Protein samples were resolved by SDS/10 % PAGE, transferred onto a nitrocellulose membrane and analyzed by Western blotting using (a) anti-039p antiserum and (b) anti-048p antiserum. In each panel, lanes 1 and 2 contain 30 mg of extract of S. solfataricus cells from logarithmic and stationary cultures, respectively. Lanes 3 and 4 of (a) and lanes 4 and 3 of (b) contain 1 mg of 039p and 048p, respectively. The position of protein markers is shown by arrows at the right and noted in kDa.

the bacterial growth curve. Therefore, it is quite likely that they are involved not only in DNA replication, but also in other important cellular functions, such as DNA repair and recombination, RNA transcription, cell cycle regulation, as recently found for eukaryotic PCNA (for a review, see Kelman, 1997). On the other hand, only one putative homolog of PCNA has been found by primary structure similarity searches in each of the other available genome sequences of Archaea (M. jannaschii, A. fulgidus, M. thermoautotrophicum, P. horikoshii; as reviewed by Edgell & Doolittle, 1997). It should be pointed out that all the above mentioned species belong to the subdomain of Euryarcheota, whereas Sulfolobus belongs to Crenarcheota. We suspect that the presence of two DNA polymerase sliding clamps is a general feature of Crenarcheota, since two ORFs coding for putative PCNA-like factors have been recently found also in the genome sequence of another thermoacidophilic crenarcheote Pyrobaculum aerophilum (Fitz-Gibbon et al., 1997; Fitz-Gibbon. T. S. and Miller, J. H., personal communication). Thus, it is tempting to speculate that the multiple DNA polymerase processivity factors found in Crenarcheota arose by an early (quite likely lineage-speci®c) gene duplication event. However, a detailed phylogenetic analysis of the PCNA-like factor sequences from these and, hopefully, other archaeal species is needed to clarify this issue. Sso DNA pol B1 could be stimulated over tenfold by 048p and about sevenfold by 039p either on poly(dA)-oligo(dT) or on linear primed singlestranded M13 DNA as a primer-template. On the other hand, a 40-fold activation by PCNA has been reported for human DNA pol d, obtained by co-

53 expression of the p125 catalytic subunit and p50 small subunit in insect cells, and this maximal stimulatory effect was obtained only in the presence of the small subunit (Zhou et al., 1997). Sso DNA pol does not seem to associate in vivo to any accessory subunit, since this enzyme was puri®ed from S. solfataricus cell extracts as a monomer (Rossi et al., 1986), and searches of the Sulfolobus genome database for homologs of eukaryotic DNA polymerase d small subunit have been unsuccessful so far. Furthermore, assembly of the DNA pol dPCNA complex at the eukaryotic replication fork requires replication factor-C, the heteropentameric clamp-loader apparatus (for a review, see Mossi & HuÈbscher, 1998). S. solfataricus, as well as other archaeal species (Edgell & Doolittle, 1997), has been found to possess putative homologs of human replication factor-C p140 and p40 subunits. The oligomeric structure and the enzymatic features of this putative clamp-loader complex from Sulfolobus are currently under investigation in our laboratory. Studying the interplay between the sliding clamp and the multiple DNA polymerase activities found in a certain organism is very helpful in order to identify the chromosomal replicase. In the eukaryotic replisome three DNA polymerases (a, d and e) have been found (Bambara et al., 1997). Whereas the role of DNA pol a in priming DNA synthesis in association with DNA primase is well documented, the exact function of DNA pols d and e remains to be elucidated and may differ between leading and lagging strands. Both DNA polymerases d and e are multisubunit enzymes which derive their high processivity from the interaction with PCNA (Baker & Bell, 1998). As for the Archaea, S. solfataricus was found to possess three family B DNA polymerases, referred to as B1 (the enzyme used in this study), B2 and B3 (Edgell et al., 1997). The DNA polymerases B2 and B3 have not been biochemically characterized. However, they should be devoid of the proof-reading function, since amino acid residues critical for the 30 -50 exonuclease activity have not been found in their primary structure. Our ®nding that S. solfataricus DNA pol B1 synthetic activity is highly enhanced in the presence of 048p (or 039p) suggests that it might be the main replicative enzyme of this organism. However, the level of stimulation by 039p seems to be appreciably lower than that effected by 048p (see Figures 4 and 6). This could depend on the fact that 039p has in vivo a biological function different from that of 048p or it could act as the sliding clamp for one of the other S. solfataricus DNA polymerases. Furthermore, since orthologs of Sso DNA pol B1 have been identi®ed in some other crenarchaeal species, including Sulfolobus acidocaldarius (Elie et al., 1989), Pyrodictium occultum (Uemori et al., 1995), P. aerophilum (FitzGibbon et al., 1997), we propose that this type of family B DNA polymerase is the main replicase of Crenarcheota.

54 On the other hand, Euryarcheota seem to possess a different set of DNA polymerase activities and it has been recently hypothesized that the chromosomal replicase of these organisms is the DNA polymerase II (Cann et al., 1998). This enzyme has been identi®ed as a novel DNA polymerase activity in some euryarchaeal species. It is composed of two subunits, DP1 (69 kDa), which is similar to the 50 kDa subunit of eukaryotic DNA pol d, and DP2 (143 kDa), which does not share any evident sequence similarity to known proteins. It would be interesting to test whether members of this novel DNA polymerase family functionally interact with the speci®c PCNA-like factors. In conclusion, all these data suggest that the molecular machineries acting at the replication fork in Crenarcheota and Euryarcheota might be different. However, development of genetic tools to speci®cally manipulate and transform archaeal cells is needed to de®nitely ascertain the biological function of these replication proteins.

Materials and Methods Materials All chemicals were reagent grade. DNA restriction and modi®cation enzymes were from Promega, unless otherwise stated. Oligonucleotides were synthesized by Primm srl (Milan, Italy). All the radioactive reagents were purchased from Amersham Life Science Products. Recombinant Sso DNA pol B1 was puri®ed as described (Pisani et al., 1998). Computer analysis of protein sequences The Sulfolobus solfataricus genome database (Institute for Marine Biosciences, NRC, Halifax, NS Canada) was searched using the BLASTP program (Altschul et al., 1990). Multiple sequence alignments were generated with the ClustalX program (version 1.64b; Jeanmougin et al., 1998). Plasmid construction Over-expression of 039p and 048p in E. coli was achieved by subcloning the corresponding genes into a pET vector derivative (pT7 SCII) under the control of the T7 RNA polymerase f 10 promoter (Studier et al., 1990). ORFs c41-039 and c41-048 were ampli®ed by polymerase chain reaction using S. solfataricus genomic DNA as the template, Expand High Fidelity Taq DNA polymerase (Boehringer Mannheim) and each of the following set of primers: 039Nde50 (50 -GGTTCCATGGCATATGAAAGTAGTTTTACGATGATGTAAGGGTT-30 ) and 039Bam30 (50 -CCTTGGATCCTCAAACTTTTGGAGCTAATAAATAAGTAACT-30 ); 048Nde50 (50 -GGTTCCATGGCATATGTTTAAGATTGTTTACCCTAATGCAAAA-30 ) and 048Bam30 (50 -CCTTGGATCCTTATAACCTTGGCGCTATCCAAAAGATCATGTGACCCCC-30 ). These oligonucleotides introduce NdeI and BamHI restriction sites upstream from the initiation codon and downstream from the stop codon of each ORF, respectively. In addition, the oligonucleotide 048Nde50 was designed to introduce a silent mutation in the triplet 240 of c41-048 ORF (CAT ! CAC) in order to eliminate a NdeI restric-

Archaeal Sliding Clamps tion site (50 -CATATG-30 ). The polymerase chain reactions were carried out for 30 cycles (one minute at 94  C, one minute at 55  C, one minute at 72  C). The ampli®cation products, eluted from gel and digested with NdeI and BamHI, were ligated into NdeI-BamHI-linearized pT7 SCII vector to create pT7-039p and pT7-048p. The cloned fragments were completely sequenced using the T7 DNA polymerase sequencing Kit (Pharmacia) to rule out that undesired mutations were introduced during ampli®cation. Expression and purification of 039p and 048p The plasmid pT7-039p (or pT7-048p) were transformed into E. coli BL 21 (DE3) cells. Transformed bacteria were grown at 37  C in one liter culture of LuriaBertani broth (supplemented with 100 mg/ml ampicillin). When the absorbance at 600 nm reached a value of 0.8, IPTG was added to a ®nal concentration of 0.5 mM to induce recombinant protein expression. After two hours at 37  C, cells were harvested by centrifugation and the bacterial pellets stored at ÿ20  C until use. The cell paste (about 4 g) was thawed and resuspended in 25 ml of buffer A (10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 100 mM EDTA, 25 mM NaCl) supplemented with some protease inhibitors (50 mg/ml PMSF, 0.2 mg/ml benzamidine, 1 mg/ml aprotinin, 10 mg/ml soybean trypsin inhibitor). Cells were broken by two consecutive passages through a French pressure cell apparatus (Aminco Co., Silver Spring, MD, USA) at 2000 psi. Bacterial lysate was clari®ed by ultracentrifugation for 15 minutes at 30,000 rpm (Sorvall rotor 50 2Ti) at 10  C. The supernatant (protein concentration: 15 mg/ml) was subjected to thermoprecipitation steps at 55 and 60  C for ®ve minutes. After each heat treatment, the sample was incubated in ice for ten minutes and the precipitated protein was removed by ultracentrifugation for 15 minutes at 30,000 rpm (Sorvall rotor 50 2Ti) at 10  C. The sample (volume: 20 ml; protein concentration: 7 mg/ml) was then chromatographed using an FPLC system (Pharmacia) through a Mono Q column (HR 10/10) equilibrated in buffer A (¯ow rate: 1 ml/min). The column was washed with buffer A and elution carried out with 50 ml linear gradient from 50 mM to 550 mM NaCl in the same buffer. Fractions (1 ml) were collected and analyzed by SDS-PAGE. Fractions containing the pure recombinant protein (centered at 0.32 M NaCl for 039p and at 0.16 M NaCl for 048p) were pooled, concentrated using the Centricon 10 system (Amicon) and dialyzed against 2  buffer A. After dialysis each protein pool (volume: 0.5 ml) was added with one volume of glycerol and stored at ÿ20  C. The ®nal yield of both the recombinant proteins puri®ed with this procedure was about 1 mg per liter of bacterial culture. NH2-terminal sequence analysis NH2-terminal sequence analysis of proteins 039p and 048p was carried out after electrotransfer onto PVDF membrane ProBlott (Applied Biosystems) from denaturing polyacrylamide gels, as described (Pisani et al., 1996). Analysis of the oligomeric state of 039p and 048p Samples of puri®ed 039p and 048p were subjected to analytical gel ®ltration chromatography on Protein Pak Glass 300 SW column (Waters) equilibrated with 50 mM sodium phosphate (pH 7.0), 100 mM NaCl buffer. The

55

Archaeal Sliding Clamps column was calibrated by running separately a set of globular protein standards. Assays for sliding clamp activity on poly(dA)-oligo(dT) The standard reaction mixture (volume 10 ml) contained 50 mM Tris-HCl (pH 8.0), 2.5 mM 2-mercaptoethanol, 2.5 mM MgCl2, 1 mM dTTP, 25 ng/ml poly(dA)/(50 -32P)-end-labeled (dT)34 (40:1 molar ratio) and the indicated amounts of 039p (or 048p). The mixtures were pre-incubated for 20 seconds at 60  C. Sso DNA pol B1 (4 ng) was then added and the incubation continued at the same temperature for an additional 30 seconds. Reactions were terminated by adding stop solution (97.5 % formamide, 10 mM EDTA (pH 7.5), 0.3 % xylene cyanol, 0.3 % bromophenol blue). Elongation products were resolved by electrophoresis in 6 % (w/v) polyacrylamide/8 M urea gels and analyzed by autoradiography or phosphorimaging (Molecular Dynamics). Activity assays on linear single-stranded M13 DNA Activity assays on linearized M13 DNA were carried out essentially as described by Burgers & Yoder (1993). A 44-mer oligonucleotide (50 - CCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAAT-30 ), designed to be complementary to single-stranded M13 mp18 DNA within the polylinker region, was utilized as a primer. The annealing reaction was carried out by incubating the DNA template mixed with this synthetic oligonucleotide (molar ratio 1:1) at 90  C for ®ve minutes. After a slow cooling at room temperature, the primed DNA was linearized by digesting it for two hours at 37  C with HincII (the restriction site is underlined in the oligonucleotide sequence above reported). Since this sample was diluted about 13-fold in the polymerase assay, carryover of restriction buffer components was negligible. The reaction mixtures (10 ml) contained 50 mM TrisHCl (pH 8.0), 2.5 mM 2-mercaptoethanol, 2.5 mM MgCl2, 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 10 mM [a-32P]dTTP (3 mCi), 60 ng (25 fmol) HincII-cut primed M13 mp18 DNA, and the indicated amounts of 039p and 048p (from 0.014 to 2.1 mg). The samples were preincubated for 20 seconds at 70  C. Sso DNA pol B1 (0.8 ng, 8 fmol) was then added and the incubation continued for 30 minutes at the same temperature. Reactions were terminated by adding one volume of 20 mM EDTA (pH 8.0), and put on ice. One half of the reaction was analyzed by alkaline agarose gel electrophoresis. The other half was precipitated by adding 0.5 ml of ice-cold 10 % (w/v) trichloroacetic acid and 10 mg of carrier calf thymus DNA. After ten minutes on ice, acid-insoluble material was collected on Whatman GF/C glass ®ber ®lter discs by ®ltration. The ®lters were extensively washed with 1 % trichloroacetic acid, and their radioactivity was determined by scintillation counting after they had been allowed to dry. Thermal stability of 039p and 048p The heat-resistance of 039p and 048p sliding clamp activity was monitored at 60 and at 80  C. Samples of each protein (140 ng in 10 ml of assay buffer containing 25 ng/ml of poly(dA)/(50 -32P)-end-labeled (dT)34) were incubated at the above temperatures in a heating block equipped with a thermostated lid to prevent evapor-

ation. At each indicated time, tubes were transferred into ice and the residual sliding clamp activity was measured on poly(dA)/oligo(dT) as a primer-template by adding to each sample Sso DNA pol B1 (4 ng) and dTTP at 1 mM, as described above. Polyclonal antibody to S. solfataricus 039p and 048p Pure 039p and 048p (200 mg) were emulsi®ed in complete Freund's adjuvant and injected subcutaneously into New Zealand rabbits. After two weeks, a booster consisting of 150 mg 039p (or 048p) in incomplete Freund's adjuvant was administered to each animal, followed by two additional injections (100 mg protein). Western blotting S. solfataricus cells were grown in 200 ml of medium by Dr A. Guagliardi and Dr L. Cerchia (University of Naples, Italy), as described (De Rosa et al., 1976). A sample of 100 ml was withdrawn from the culture when the A600 nm reached the value of 0.6 (logarithmic phase). Cells were then pelleted by centrifugation for ten minutes at 8000 rpm (Sorvall GSA rotor) and stored at ÿ20  C until use. The remaining portion of the culture was grown up to stationary phase (A600 nm ˆ 1.5). Cells were then harvested by centrifugation for ten minutes at 8000 rpm (Sorvall GSA rotor) and stored at ÿ20  C until use. Both pellets were thawed and resuspended in buffer 50 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 2 mM 2-mercaptoethanol. Cells were disrupted by sonication on ice and the extract clari®ed by centrifugation for 15 minutes at 30,000 rpm (Sorvall rotor 50 2Ti). For immunoblot analysis SDS-10 % polyacrylamide gels were electroblotted onto nitrocellulose membranes (Schleicher & Schuell). The blots were blocked with 3 % (w/v) bovine serum albumin (Fraction V, Sigma) in Trisbuffered saline (TBS) for 30 minutes, and then incubated for over two hours at room temperature with a 1:1000 dilution of rabbit anti-039p (or anti-048p) antiserum in 3 % bovine serum albumin in TBS containing 0.05 % (v/v) Tween-20 (TBS-T). Membranes were washed using TBS-T and then incubated for one hour at room temperature with a 1:2000 dilution of horseradish peroxidaseconjugated rabbit anti-rabbit IgG (Bio-Rad Labs) in 3 % bovine serum albumin in TBS-T. Membranes were washed using TBS-T and the secondary antibody detected with the standard colorimetric procedure, according to manifacturer's instructions.

Acknowledgements Dr Pierangelo Orlando is gratefully aknowledged for helpful discussions and suggestions. This work received ®nancial support in the form of grants from Consiglio Nazionale delle Ricerche (Progetto Finalizzato Biotecnologie) and from the European Union (Contract B 104-CT96-0488) to M. R. This is NRCC publication no. 42298.

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Edited by J. H. Miller (Received 5 February 1999; received in revised form 2 June 1999; accepted 4 June 1999)