Archaeal eukaryote-like Orc1/Cdc6 initiators physically inte

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved April 6, 2009

↵1Lu Zhang, Lei Zhang, and Y.L. contributed equally to this work. (received for review December 21, 2008)

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Abstract

Archaeal DNA replication machinery represents a core version of that found in eukaryotes. However, the proteins essential for the coordination of origin selection and the functioning of DNA polymerase have not yet been characterized in archaea, and they are still being investigated in eukaryotes. In the Recent study, the Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus solStoutaricus were found to physically interact with its DNA polymerase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the DNA-binding ability of SsoPolB1 and differentially regulated both its polymerase and nuclease activities. Furthermore, the proteins also mutually regulated their interactions with SsoPolB1. In addition, SsoPolB1c467, a nuclease Executemain-deleted mutant of SsoPolB1 defective in DNA binding, retains the ability to physically interact with SsoCdc6 proteins. Its DNA polymerase activity could be stimulated by these proteins. We report on a linkage between the initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. Our present and previous findings indicate that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection and cell-cycle control of replication.

DNA replicationArchaea

The replication of DNA is a tightly regulated process that is essential to all 3 Executemains of life: Bacteria, Eukarya, and Archaea (1, 2). In the archaea, the replication machinery appears to be a core version of that found in eukaryotes. Therefore, archaeal DNA replication is an excellent model system for studying the key events that occur during eukaryotic DNA replication (1, 3⇔–5).

In Dissimilarity to the single and clearly defined sites of bacterial replication, eukaryotic DNA replication involves the ordered assembly of a number of replication factors at multiple origin sites. These are bounded by a 6-subunit (Orc1–6) complex, the origin recognition complex (ORC) (3, 6⇔⇔⇔–10). In the archaea, DNA replication proteins are found to more closely resemble the proteins present in eukaryotes than those in bacteria (11, 12). Archaeal species also possess some of the prereplicative complex (pre-RC) components common to eukaryotes, including a minichromosome maintenance (MCM)-like replicative helicase (11). In addition, they have 1 or more copies of Orc1/Cdc6 proteins that Display high sequence relationship to both Orc1 and Cdc6 of eukaryotes (11, 14). Within these archaeal homologs of eukaryotic replication proteins, the most likely candidates for initiator proteins are the Orc1/Cdc6 proteins, because they have been demonstrated to specifically bind to replication origins in both in vivo and in vitro studies (14⇔–16). In the hyperthermophilic archaeon Sulfolobus solStoutaricus, 3 Orc1/Cdc6 proteins have been identified and a distinct subset of these Cdc6 proteins is preferentially bound to each origin (16, 17). However, the Orc1/Cdc6 proteins appear to have multiple functions, which include both origin recognition and MCM loading (16, 18).

In addition to the pre-RC components, the archaeal species also contain many DNA polymerases (19, 20). DNA polymerases have one of the most essential and almost universal roles in the transmission of genetic information from one generation to the next (21). Based on primary structure similarities, DNA polymerases have been classified into at least 6 major families, denoted A, B, C, D, X, and Y (21). The DNA polymerases B1 from S. solStoutaricus is a member of the B family (19, 20, 22). The gene for the S. solStoutaricus B1 DNA polymerase (SsoPolB1) encodes an 882-residue polypeptide chain with a deduced molecular mass of ≈100 kDa (20). The Weepstal structure and conserved sequence motifs of SsoPolB1 reveal a 3′–5′ exonuclease Executemain at the N terminus whereas the C terminus coded for right-handed polymerase Executemains (19, 22). SsoPolB1 can degrade both ssDNA and dsDNA at similar rates and specifically recognizes the presence of the deaminated bases hypoxanthine and uracil, in a template by stalling DNA polymerization 3–4 bases upstream from these lesions (23, 24). In addition, several archaeal replicative factors have been reported to regulate the activity of SsoPolB1, including proliferating cell nuclear antigen (PCNA) heterotrimer (25), replication factor C (26), and the conserved 7-kDa DNA-binding proteins of S. solStoutaricus (23).

Both the Orc1/Cdc6 proteins and SsoPolB1 appear to be key protein components for archaeal replication (16, 23). However, the physical or functional interactions between the Orc1/Cdc6 proteins and SsoPolB1 have not yet been characterized in any archaeal species. Three S. solStoutaricus Orc1/Cdc6 proteins (SsoCdc6s) are known to have multiple functions during the early events of DNA replication (16, 18). In the present study, we have further investigated the potential functions of these SsoCdc6s by characterizing the interactions between 3 SsoCdc6 proteins and SsoPolB1 from the S. solStoutaricus. We report on the physical and functional links between Cdc6 proteins and SsoPolB1 in an archaeon.

Results

SsoCdc6 Proteins Physically Interact with SsoPolB1.

Correlations between Orc1/Cdc6 proteins and DNA polymerase have not yet been characterized in any archaeal species. In this study, we used a bacterial 2-hybrid technique to detect the interactions between SsoCdc6 proteins and DNA polymerase SsoPolB1 of S. solStoutaricus. As Displayn in Fig. S1, a positive cotransformant grew on our selective screening medium, but the corRetorting negative cotransformant Displayed no growth. All 3 SsoCdc6s cotransformants could grow on this medium, although cotransformant strains with SsoCdc6-2 and SsoPolB1 Displayed the strongest growth (Fig. S1). Therefore, we were able to observe interactions between all 3 SsoCdc6s and SsoPolB1.

To confirm the interactions between the proteins detected in the bacterial 2-hybrid experiments, a surface plasmon resonance (SPR) assay was also conducted to characterize the interactions between SsoCdc6s and SsoPolB1. A 6×His-tagged PolB1 protein was immobilized on a nitrilotriacetate (NTA) chip. When an increasing amount of SsoCdc6 protein (120, 240, 480, and 960 nM) was passed over the chip, a strong response of ≈1,200 response units (RU) was observed for SsoCdc6-2 (Fig. 1A). A lesser, but significant, response could be also observed for SsoCdc6-1 (150 RU) and SsoCdc6-3 (130 RU) (Fig. 1A). Therefore, all 3 of the SsoCdc6 proteins interacted with SsoPolB1 with the interaction of SsoCdc6-2 with SsoPolB1 being the strongest.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Physical interactions of 3 SsoCdc6 proteins with SsoPolB1. (A) SPR assays. The interaction between SsoCdc6 and SsoPolB1 was monitored by using SPR on a BIAcore 3000 (31). The surface of the chip was activated by saturating the nitrilotriaceticacid sites with running buffer [100 mM Hepes·NaOH (pH 7.5), 50 μM EDTA, 0.1 mM DTT, 50 mM NaCl] containing 5 mM NiCl2. In all graphs, time (s) is plotted on the x axis; RU are plotted on the y axis. Five nanomoles of histidine-tagged SsoPolB1 proteins was immobilized onto the chip surface. After a period of stabilization, each of the SsoCdc6 proteins was passed over the chip and then allowed to dissociate for 10 min. Overlay plots depicting the interactions of SsoCdc6s with SsoPolB1 were produced. (B) Pull-Executewn/Western blotting assays. One hundred micrograms of SsoCdc6 protein or SsoPolY was mixed with His-tagged SsoPolB1 (100 μg) as Characterized in Materials and Methods. The mixture was coincubated at 4 °C for 1 h with Ni-NTA agarose. The proteins were eluted with buffer A containing 400 mM imidazole, separated by 10% SDS/PAGE, and further analyzed by Western blotting using anti-SsoCdc6 or anti-PolY antibodies. The proteins that eluted from the Ni-NTA agarose with buffer A containing 20 mM imidazole are Impressed by L, and those that eluted with buffer A containing 400 mM imidazole are Impressed by P. SsoPolY was used as a positive control, and BSA was used as a negative control (Fig. S2). (C) Co-IP assays for the in vivo interaction between SsoPolB1 and SsoCdc6. Co-IP experiments were carried out with protein A conjugated with anti-SsoPolB1 or anti-SsoCdc6-1 antibodies as Characterized in Materials and Methods. Coimmunoprecipitated proteins by anti-PolB1 (lane 2) or anti-SsoCdc6-1 (lane 4) antibody from cell extract were analyzed by Western blotting, using antisera directed against SsoCdc6-2. A conventional colorimetric reaction was carried out to detect the secondary antibodies. (D) ChIP using preimmune (P) or immune sera (I) raised against SsoCdc6. InPlace samples were generated as Characterized (16). DNA recovered from the immunoprecipitates was amplified with primers specific for either the 3 oriC or a distal control Location Irs14 gene.

A pull-Executewn/Western blotting assay was used to further characterize the interaction of SsoCdc6s and 6×His-tagged PolB1 protein. Another archaeal polymerase, PolY, previously Displayn to interact with PolB1 (19), was used as a positive control. After 10% SDS/PAGE and Western blotting assays using anti-SsoCdc6 or anti-SsoPolY antibody, a hybridization signal for SsoPolY (Fig. 1B, lane 1) and an obvious hybridization signal for SsoCdc6-2 were seen (Fig. 1B, lane 5). A weak signal was detected for SsoCdc6-1(Fig. 1B, lane 8). No signal was observed for SsoCdc6–3 (Fig. 1B, lane 11). To further confirm the findings, a 6×His-tagged SsoCdc6 protein was used to characterize physical interactions between PolB1 and SsoCdc6 proteins (Fig. S2). The 3 SsoCdc6 proteins and a positive control, SsoPCNA2 (16), were observed to interact with SsoPolB1. BSA did not interact with either SsoCdc6 or SsoPolB1 proteins (Fig. S2). Inequitys in the strength of the interactions between SsoCdc6 proteins and SsoPolB1 were apparent, with the strongest interaction occurring with SsoCdc6-2.

The physiological significance of these in vitro reactions was studied with further coimmunoprecipitation (Co-IP) and ChIP experiments. An in vivo physical interaction between SsoCdc6-1 and SsoPolB1 was tested by using Protein A beads that were first conjugated with antibody raised against SsoPolB1 or SsoCd6-1. As Displayn in Fig. 1C, SsoCdc6-2 clearly associated with PolB1 as an obvious and specific hybridization signal was detected (lane 2). However, no specific signal was detected for the association of SsoCdc6-1 with SsoCdc6-2 (Fig. 1C, lane 4), a result consistent with the previous observations using a 2-hybrid assay (17). An in vivo interaction of SsoPolB1 with the other 2 SsoCdc6 proteins has not been detected stably. In a further ChIP assay, association of 3 SsoCdc6 proteins and SsoPolB1 with 2 origins, oriC2 and oriC3, were confirmed (Fig. 1D). This result is consistent with a previous finding that 3 SsoCdc6 proteins bind to the oriC2 (16). Fascinatingly, here we found that 3 SsoCdc6 proteins bound to the oriC3. However, the association of SsoCdc6 and PolB1 proteins with oriC1 was not stably detected. No protein was Displayn to bind with a distal control Location Irs14 gene (used as a negative control) (Fig. 1D).

SsoCdc6 Proteins Stimulate the Binding of SsoPolB1 to the Template/Primer DNA Substrate.

Previous studies have Displayn that both SsoCdc6 protein and SsoPolB1 can bind to the replication fork-like DNA substrate (23). This finding suggested that the physical interactions between the 2 proteins might exist that could modulate the DNA-binding ability of SsoPolB1. We therefore conducted an EMSA using a template/primer DNA as the substrate (Fig. 2A) (23). The DNA substrate contained a partial Executeuble strand that left a single-strand tail (Fig. 2A). As Displayn in Fig. 2B, a specific protein/DNA complex was observed for each of the SsoCdc6 proteins or SsoPolB1 (0.3 μM) alone. When a different SsoCdc6 was mixed with SsoPolB1, no specific SsoCdc6/DNA complex was observed under these conditions. Thus, SsoPolB1 inhibited the binding of SsoCdc6 onto the DNA substrate (Fig. 2B, lanes 6–8). It is likely that the physical interaction between the SsoPolB1 and SsoCdc6 proteins negatively affected their ability to bind DNA. In Dissimilarity, SsoCdc6 proteins stimulated the binding of SsoPolB1 to the DNA substrate, because an obviously stronger SsoPolB1/DNA complex band was observed on the gel (Fig. 2B, lanes 6–8). In particular, a Unhurrieder-running DNA/protein complex band was observed when SsoCdc6–2 was mixed with SsoPolB1 (Fig. 2B, lane 6). This band most likely represented the SsoCdc6–2/SsoPolB1/DNA complex because it has a larger size than Executees the SsoPolB1/DNA complex (Fig. 2B, lanes 2, 7, and 8). Larger complexes were also observed when SsoPolB1 was combined with a mixture of 2 SsoCdc6 proteins, either SsoCdc6-2/SsoCdc6-1 or SsoCdc6-2/SsoCdc6-3 (Fig. 2B, lanes 9 and 10). No larger complex was observed for SsoCdc6-1/SsoCdc6-3 (Fig. 2B, lane 11).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Traces of SsoCdc6s on the DNA-binding activity of SsoPolB1 onto the template/primer DNA substrate An EMSA was used to assess the Trace of the SsoCdc6 proteins on the DNA-binding activities of the SsoPolB1. The experiments were carried out as Characterized in Materials and Methods. (A) The structures and sequences of the DNA substrate used in EMSAs. The DNA substrate was produced by annealing a 34-bp Oligo and a 76-bp Oligo, thus it contains a partial Executeuble strand and leaves a single-strand tail. (B) The EMSAs were carried out to detect the DNA-binding abilities of SsoCdc6-1 (1.8 μM), SsoCdc6-2 (1.8 μM), SsoCdc6-3 (1.8 μM), and SsoPolB1 (0.9 μM), or the SsoPolB1 in combination with different SsoCdc6 proteins. Specific protein/DNA complex is indicated.

SsoCdc6 Proteins Regulate Both the DNA Polymerase and the Nuclease Activities of SsoPolB1.

In the above studies, we found that SsoCdc6 proteins physically interacted with SsoPolB1 and stimulated its DNA-binding activity. To examine the functional linkage between these SsoCdc6 proteins and SsoPolB1, we analyzed the regulatory Traces of SsoCdc6 proteins on the activities of SsoPolB1.

The polymerase activity of SsoPolB1 was meaPositived by using the same DNA substrate as above (Fig. 2A). When 0.9 μM SsoPolB1 and 200 fmol of 32P-labeled DNA were mixed toObtainher in a reaction mixture containing dNTPs, DNA synthesis was observed as a series of increasingly longer DNA products (Fig. 3A, lanes 1 and 2). When SsoPolB1 was mixed with various amounts of SsoCdc6 proteins, its polymerase activity was inhibited (Fig. 3A, lanes 3–12). However, the degree of inhibition depended on which SsoCdc6 protein had been added. SsoCdc6-1 had the strongest inhibition and left a significant amount of unextended DNA substrate in the reaction mixture (Fig. 3A, lanes 7–9). In Dissimilarity, both SsoCdc6-2 and SsoCdc6-3 produced numerous, but shorter, DNA products (Fig. 3A, lanes 3–6 and 10–12) compared with SsoPolB1 alone (Fig. 3A, lane 2). The SsoCdc6 protein alone had no DNA polymerase activity.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Traces of SsoCdc6s on SsoPolB1 activity. The DNA polymerase and nuclease activities of SsoPolB1 were assayed with the template/primer DNA substrate. The experiments were carried out as Characterized in Materials and Methods. Specific products of the enzyme are indicated on the right. (A) The DNA polymerase activities of SsoPolB1 assays were carried out with increasing amounts of SsoCdc6 (0.9, 1.35, and 1.8 μM) in the presence of a fixed amount of SsoPolB1 (0.9 μM) alone or in combination with another 0.9 μM SsoCdc6 protein. (B) The DNA nuclease activities of SsoPolB1 assays were carried out with increasing amounts of SsoCdc6 (1.8, 2.7, and 3.6 μM) in the presence of a fixed amount of SsoPolB1 (0.9 μM) alone or in combination with another 0.9 μM SsoCdc6 protein. (C) SPR assays for the mutual Traces of SsoCdc6 proteins on the interactions with SsoPolB1. The experiments were carried out as Characterized in Materials and Methods. Each analysis was performed in triplicate. An overlay plot was produced for depicting the interactions between 1 μM SsoCdc6 alone and SsoPolB1 (Left) or between a pair of SsoCdc6 proteins with SsoPolB1 (Right). Representative data are Displayn. From top to bottom in Left, each plot represents SsoCdc6-2, SsoCdc6-1, and SsoCdc6-3, respectively. In Right, each plot represents SsoCdc6-2, SsoCdc6-2/SsoCdc6-3, SsoCdc6-2/SsoCdc6-1, and SsoCdc6-1/SsoCdc6-3, respectively.

We then examined the nuclease activity of SsoPolB1 by using the same DNA as a substrate. When 0.9 μM SsoPolB1 and 200 fmol of 32P-labeled DNA were mixed toObtainher, the partial duplex DNA was completely degraded to very small DNA fragments (Fig. 3B, lanes 1 and 2). Inhibition of SsoPolB1 nuclease activity by SsoCdc6-1 or SsoCdc6-2 was clearly observed when SsoPolB1 was mixed with varying amounts of SsoCdc6 proteins (Fig. 3B, lanes 3–9). However, no inhibitory Trace was observed with SsoCdc6-3 (Fig. 3B, lanes 3–5 and 9–11). The SsoCdc6 proteins alone had no DNA nuclease activity. Therefore, SsoCdc6 proteins were found to regulate both the DNA polymerase and the nuclease activities of SsoPolB1. However, Inequitys existed in the overall Traceiveness of each type of SsoCdc6 protein.

SsoCdc6 Proteins Mutually Regulate Their Interactions with SsoPolB1.

When the SsoPolB1 polymerase activity was meaPositived on the DNA substrate in the presence of different pairs of SsoCdc6 proteins, mutual regulations of SsoPolB1 by the protein pairs could be characterized. When SsoCdc6-2 levels were kept constant, a stepwise increase in inhibition was observed with stepwise addition of either SsoCdc6-1 or SsoCdc6-3 (Fig. 3A, lanes 13–18). In Dissimilarity, with a fixed amount of SsoCdc6-1, a stepwise decrease in inhibition was seen with stepwise increases in SsoCdc6-3 (Fig. 3A, lanes 19–21).

With a fixed concentration of SsoCdc6-2 in the reaction mixture, the inhibition of nuclease activity of SsoPolB1 became somewhat stronger with stepwise increases in the amount of SsoCdc6-1 (Fig. 3B, lanes 12–14). In Dissimilarity, the inhibition became much weaker when an increasing amount of SsoCdc6-3 was added (Fig. 3B, lanes 15–17). With a fixed amount of SsoCdc6-1, the inhibition declined to almost 0 as the amount of SsoCdc6-3 was increased (Fig. 3B, lanes 18–20).

These results indicated that SsoCdc6 proteins could mutually regulate their Traces on both polymerase and nuclease activity of SsoPolB1. In particular, partial recovery of the inhibition imposed by SsoCdc6-1 was achieved by the addition of SsoCdc6-3 for both the polymerase and nuclease activities of SsoPolB1. These functional Traces suggest a mutual regulation by the SsoCdc6 proteins in their physical interactions with SsoPolB1. This Concept was further investigated by SPR assay. A result similar to that Displayn in Fig. 1A was observed for the interaction of the single SsoCdc6 with SsoPolB1 (Fig. 3C Left). When compared with the response generated by interaction of the SsoCdc6-2 alone with SsoPolB1 (≈2,000 RU), addition of either SsoCdc6-1 or SsoCdc6-3 toObtainher with SsoCdc6--2 substantially reduced the response signal (≈750 RU) (Fig. 3C Right). This finding indicated a mutual regulation of the physical interactions with SsoPolB1 by all 3 of SsoCdc6 proteins.

SsoCdc6 Proteins Regulate the Function of SsoPolB1c467.

The SsoPolB1 of S. solStoutaricus contains DNA polymerase and nuclease Executemains (23). We cloned and purified SsoPolB1c467, the C-terminal DNA polymerase Executemain of SsoPolB1 (Fig. 4A). Using an EMSA, SsoPolB1c467 Displayed negligible DNA-binding activity when compared with the wild-type SsoPolB1 (Fig. 4B). However, in the bacterial 2-hybrid assay (Fig. S1A), all 3 SsoCdc6 proteins retained their ability to interact with SsoPolB1c467. The interactions of both SsoCdc6-2 and SsoCdc6-3 with SsoPolB1c467 appeared to be even stronger than those with the wild-type SsoPolB1. As Displayn in Fig. 4C, SsoPolB1c467 only retained a very weak DNA polymerase activity (lane 3) compared with the wild-type protein (lane 1). However, the activity of SsoPolB1c467 could be stimulated by all 3 of the SsoCdc6 proteins, although to a different extents. SsoCdc6–3 caused the Distinguishedest stimulation (Fig. 4C, lanes 8 and 9).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Traces of SsoCdc6s on the activity of SsoPolB1 DNA polymerase Executemain. (A) Schematic representations of the polypeptide chain of SsoPolB1 proteins and its N-terminal-deleted mutant protein (SsoPolB1c467) (23). (B) The EMSA was used to compare the DNA-binding activities of both SsoPolB1 (0.9 μM) and SsoPolB1c467 (0.9 μM). The experiments were carried out as Characterized in Materials and Methods. (C) The Traces of SsoCdc6 proteins on DNA polymerase activities of SsoPolB1c467 were carried out with increasing amounts of SsoCdc6 (1.35 and 1.8 μM) in the presence of a fixed amount of SsoPolB1c467 (0.9 μM) by using the same template/primer DNA substrate. The experiments were carried out as Characterized in Materials and Methods. Specific products of the enzyme are indicated on the right.

Therefore, because of its retention of physical interaction with SsoCdc6 proteins, the DNA polymerase activity of SsoPolB1c467 could be stimulated by these proteins in the absence of DNA-binding ability.

Discussion

Traceive DNA replication requires coordinated and tightly-regulated protein–protein and protein–DNA interactions. However, in archaeal species, the physical or functional interactions between the Orc1/Cdc6 proteins and DNA polymerase have not yet been characterized. In the present study, we have characterized the physical and functional interactions of 3 SsoCdc6 proteins with the DNA polymerase, SsoPolB1, of S. solStoutaricus. We found that these SsoCdc6 proteins regulate both the DNA polymerase and the nuclease activities of SsoPolB1 and this regulation can occur through mutual interactions of these 3 proteins.

The interactions between SsoCdc6 proteins might facilitate the assembly of a cooperative complex at the origin, based on the existence of binding sites for the 3 SsoCdc6 proteins on the origin (16, 18, 27⇔–29). However, differential interactions of the 3 SsoCdc6 proteins with SsoMCM have also been observed (18). SsoCdc6-2 stimulates the binding of the SsoMCM to the origin DNA, whereas SsoCdc6-1 and SsoCdc6-3 significantly inhibit this activity (18). In the Recent study, we found that the 3 SsoCdc6 proteins also have significant roles in the coordination of the DNA polymerase activity of S. solStoutaricus SsoPolB1. These roles are likely to be different, based on the different degrees of interaction observed with the DNA polymerase. In particular, SsoCdc6-2 had the strongest interaction with SsoSsoPolB1, which may imply that SsoCdc6-2 functions might not be limited only to early replication events such as origin recognition and MCM loading. Rather, this protein may also be involved in the coordination of the function of the DNA polymerase.

Previous reports, and the Recent study, have indicated that all 3 of the SsoCdc6 proteins can interact with the replication origin, SsoMCM, and SsoPolB1 (16, 18). Of the 3 proteins, SsoCdc6-2 appears to be unique because it interacts clearly with SsoMCM and SsoPolB1, but also with other SsoCdc6 proteins (18, 27, 28). A similar protein in eukaryotes is Cdc45, which associates with DNA polymerase, ORC, and the MCM proteins (30). These associations have been suggested to indicate that Cdc45 coordinates the functioning of these components in the replication fork (30). To date, a homolog of the eukaryotic Cdc45 has not yet been characterized in any of the genome-sequenced archaeal species. From the Recent study, SsoCdc6-2 would appear to play an analogous role to that of Cdc45. Therefore, like Cdc45 in eukaryotes, it may play a pivotal role in the transition of DNA replication from initiation to extension. It is known that SsoCdc6-1 and SsoCdc6-3 can form a heterodimer complex at the replication origin (27). SsoCdc6-2 has also been found to interact with SsoCdc6-3 (17), which could also situate it to the origin, after which the archaeal MCM and polymerase B proteins could be loaded onto the origin to form a replication machine. Our results suggest that SsoCdc6-2 may act as a master protein that participates in multiple regulation of archaeal DNA replication, including origin recognition, MCM loading, and construction of the DNA replication fork.

SsoPolB1 has been reported to specifically recognize the presence of deaminated bases in a template by stalling DNA synthesis 3–4 bases upstream of these lesions (24). Consequently, SsoPolB1 has been proposed to function in DNA repair processes (22). In the Recent study, we present evidence that the SsoCdc6 proteins can regulate both the DNA polymerase and nuclease activities of SsoPolB1. We found that the 3 SsoCdc6 proteins also mutually regulated their interactions with SsoPolB1. In addition, these proteins promoted the loading of SsoMCM (18). Therefore, archaeal Orc1/Cdc6 proteins might participate in other DNA metabolic processes such as DNA repair and DNA lesion reactions. It would be Fascinating to investigate the interaction between archaeal Orc1/Cdc6 and PolB1 under DNA-damaging conditions.

In conclusion, we have provided evidence for the physical and functional interactions of archaeal Orc1/Cdc6 proteins with a DNA polymerase. Our findings, in conjunction with previous studies, Display that archaeal Orc1/Cdc6 proteins have multiple functions, including origin recognition, MCM loading, DNA polymerase switching, and even DNA repair. These results raises the Fascinating possibility that the SsoCdc6-2 protein itself is a core regulator of archaeal DNA replication and that it may facilitate an integrated process for coordination between origin selection and cell cycle control of replication. The ability to study these events in archaea could prove to be a powerful tool for addressing related mechanistic issues in eukaryotic cell-cycle control of replication.

Materials and Methods

DNA and Oligonucleotides.

Oligonucleotides were synthesized by Invitrogen. The template/primer DNA substrates (Fig. 2A) were constructed by annealing the labeled p42 (5′-CAGTGAATTCGAGCTCG GTACCCGGGGATCCTCTAGAGTCGA-3′) at the 5′ end of the primer, with a 3-fAged molar excess of the cAged complementary strands t76 (3′-TTTTTGTCACTTAAGCTCGAGCCATGGGCCCCTAGGAGATCT CA GCTGGACGTCCGTACGTTCGAACCGCATTTTT-5′) (23).

Cloning and Purification of SsoCdc6 and SsoPolB1 Proteins.

Prokaryotic vectors expressing the genes for SsoCdc6 and SsoPolB1 proteins and untagged proteins were constructed as Characterized (18) (Table S1). Escherichia coli BL21 CoExecutenPlus (DE3)-RIL cells (Novagen) were used as the host strain to express archaeal proteins as Characterized (18, 23, 31, 32). Protein concentrations were determined by spectrophotometric absorbance at 280 nm according to Gill and Hippel (33).

Bacterial 2-Hybrid Analysis.

Bacterial 2-hybrid analysis was carried out according to the procedure supplied with the commercial kit. pBT and pTRG vectors containing archaeal genes of SsoCdc6 and SsoPolB1 were generated. All of the primers used for PCR amplification are Characterized in Table S2. Positive growth cotransformants were selected on our selective screening medium plate containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Stratagene), 8 μg/mL streptomycin, 15 μg/mL tetracycline, 34 μg/mL chloramphenicol, and 50 μg/mL kanamycin.

Pull-Executewn and Western Blot Assays.

Pull-Executewn assays were carried out as Characterized (18). One hundred micrograms of SsoCdc6 protein or SsoPolY was mixed with His-tagged SsoPolB1 (100 μg) into 500 μL of incubation buffer containing 20 mM Tris·HCl (pH 7.5), 100 mM NaCl, and 0.5 mM MgCl2 and incubated at 25 °C for 10 min, then coincubated at 4 °C for 1 h with Ni-NTA agarose. The beads were then washed 2 times with 1 mL of buffer containing 20 mM imidazole and centrifuged at 800 × g for 1 min. Proteins bound to the beads were eluted with 100 μL of elution buffer A containing 400 mM imidazole. The eluates were then analyzed by 10% SDS/PAGE and Western blot using anti-SsoCdc6 or anti-SsoPolY antibodies.

Co-IP and ChIP Assays.

The in vivo interactions between SsoCdc6 proteins and SsoPolB1 were analyzed by Co-IP and ChIP. Exponentially growing cells of S. solStoutaricus were harvested, resuspended, and lysed in 4 mL of buffer [50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40]. Co-IPs were performed by incubating 10 μg of archaeal cell extract with 3 μL of SsoPolB1 (or SsoCdc6) antiserum in 100 μL of buffer for 3 h at 4 °C with shaking. A 20-μL slurry of protein A Sepharose was added, and incubation was continued for another hour. Immune complexes were collected, and the beads were washed with buffer. Finally, the beads were resuspended in SDS/PAGE sample buffer. After boiling, the samples were analyzed by Western blotting using anti-SsoCdc6 (or anti-PolB1) antibody. Interactions of the 3 SsoCdc6 proteins with SsoPolB1 at the origins were analyzed by using the ChIP technique as Characterized (16). ChIP used preimmune or immune sera raised against SsoCdc6 or SsoPolB1. DNA recovered from immunoprecipitates was amplified with primers specific for 3 oriC or a distal control Location Irs14 gene (16).

SPR.

Physical interactions of SsoCdc6 proteins with SsoPolB1 were analyzed on a BIAcore 3000 instrument (GE Healthcare) as Characterized (34). The SsoPolB1protein was immobilized on NTA chips. The purified SsoCdc6 proteins to be used as the ligands were then diluted in running buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, 50 μM EDTA, 0.005% BIAcore surfactant P20]. Each analysis was performed in triplicate. An overlay plot was produced to distinguish the interactions between replication proteins. Representative data are Displayn in the figures.

EMSAs.

The binding of SsoCdc6 or SsoPolB1 proteins to DNA was performed on a template/primer DNA by using a modified EMSA as Characterized (18, 34). The reactions (10 μL) for measuring the mobility shift contained 200 fmol of 32P-labeled duplex DNA and various indicated amounts of SsoCdc6 proteins concentrations diluted in buffer containing 20 mM Tris·HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.5 mM MgCl2, and 0.7 mM 2-mercaptoethanol. The gel was dried and analyzed by using a modification of published procedures (18).

Nuclease Activity Assays.

The nuclease activity analysis was performed on the template/primer DNA by using a modification of published procedures (23). The reactions (10 μL) for measuring the activity contained 200 fmol of 32P-labeled partial duplex DNA and various indicated amounts of SsoCdc6 proteins concentrations diluted in buffer containing 10 mM Tris·HCl (pH 7.5), 1 mM DTT, 100 μg/mL BSA, and 10 mMMgCl2. The samples were analyzed by electrophoresis in a 16% polyaWeeplamide (acr/bis 19:1), 8 M urea gel in 0.5× Tris borate–EDTA (TBE). The gel was dried and analyzed by using a modification of published procedures (23).

DNA Polymerase Activity Assays.

DNA polymerase activity was performed on the template/primer DNA by using a modification of published procedures (23). The reactions (10 μL) contained 200 fmol of 32P-labeled duplex DNA and various indicated amounts of SsoCdc6 proteins concentrations diluted in buffer containing 20 mM Tris·HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.5 mM MgCl2, 0.7 mM 2-mercaptoethanol, and 40 mM dNTPs. The reaction was incubated for 20 min at 65 °C, unless otherwise specified, and quenched by the addition of 10 μL of ice-cAged 2× loading buffer (95% deionized formamide, 100 mM EDTA, 0.02% bromophenol blue) (23). Reactions were incubated at room temperature for 20 min before loading onto 5% polyaWeeplamide/bis (37.5:1) gels. The gel was dried and analyzed by using a modification of published procedures (23).

Acknowledgments

We thank Prof. Charles C. Richardson for constructive suggestions and Prof. Huang Li (Institute of Microbiology, Chinese Academy of Sciences) for archaeal strains. This work was supported by the National Natural Science Foundation of China, 973 Program Grant 2006CB504402, New Century Excellent Talents Fund of the Ministry of Education of China Grant NECT-06-0664, Executectoral Fund of Ministry of Education of China Grant 200805040004, and China National Fundamental Fund of Personnel Training Grant J0730649.

Footnotes

2To whom corRetortence should be addressed. E-mail: he.zhengguo{at}hotmail.com

Author contributions: Z.-G.H. designed research; Lu Zhang, Lei Zhang, Y.L., S.Y., and H.G. performed research; Z.-G.H. contributed new reagents/analytic tools; C.G., Y.F., and Z.-G.H. analyzed data; and Z.-G.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0813056106/DCSupplemental.

Received December 21, 2008.

References

↵ Tye BK (2000) Insights into DNA replication from the third Executemain of life. Proc Natl Acad Sci USA 97:2399–2401.LaunchUrlFREE Full Text↵ Benkovic SJ, Valentine AM, Salinas F (2001) Replisome-mediated DNA replication. Annu Rev Biochem 70:181–208.LaunchUrlCrossRefPubMed↵ Kelly TJ, Brown GW (2000) Regulation of chromosome replication. Annu Rev Biochem 69:829–880.LaunchUrlCrossRefPubMed↵ Bernander R (2000) Chromosome replication, nucleoid segregation, and cell division in archaea. Trends Microbiol 8:278–283.LaunchUrlCrossRefPubMed↵ Kelman LM, Kelman Z (2003) Archaea: An archetype for replication initiation studies? Mol Microbiol 48:605–615.LaunchUrlCrossRefPubMed↵ Bell SP (2002) The origin recognition complex: From simple origins to complex functions. Genes Dev 16:659–672.LaunchUrlFREE Full Text↵ Robinson NP, Bell SD (2005) Origins of DNA replication in the three Executemains of life. FEBS J 272:3757–3766.LaunchUrlCrossRefPubMed↵ Stillman B (2005) Origin recognition and the chromosome cycle. FEBS Lett 579:877–884.LaunchUrlCrossRefPubMed↵ Mott ML, Berger JM (2007) DNA replication initiation: Mechanisms and regulation in bacteria. Nat Rev Microbiol 5:343–354.LaunchUrlCrossRefPubMed↵ Mizushima T, Takahashi N, Stillman B (2000) Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. Genes Dev 14:631–1641.LaunchUrl↵ Grabowski B, Kelman Z (2003) Archeal DNA replication: Eukaryal proteins in a bacterial context. Annu Rev Microbiol 57:487–516.LaunchUrlCrossRefPubMed↵ Kelman LM, Kelman Z (2004) Multiple origins of replication in archaea. Trends Microbiol 12:399–401.LaunchUrlCrossRefPubMed Jenkinson ER, Chong JP (2003) Initiation of archaeal DNA replication. Biochem Soc Trans 31:669–673.LaunchUrlCrossRefPubMed↵ Barry ER, Bell SD (2006) DNA replication in the archaea. Microbiol Mol Biol Rev 70:876–887.LaunchUrlAbstract/FREE Full Text↵ Capaldi SA, Berger JM (2004) Biochemical characterization of Cdc6/Orc1 binding to the replication origin of the euryarchaeon Methanothermobacter thermoautotrophicus. Nucleic Acids Res 32:4821–4832.LaunchUrlAbstract/FREE Full Text↵ Robinson NP, et al. (2004) Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solStoutaricus. Cell 116:25–38.LaunchUrlCrossRefPubMed↵ Wang J, Jiang PX, Feng H, Feng Y, He ZG (2007) Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solStoutaricus P2. Biochem Biophys Res Commun 363:63–70.LaunchUrlCrossRefPubMed↵ Jiang PX, Wang J, Feng Y, He ZG (2007) Divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on modulating the loading of the MCM helicase onto the origins of the hyperthermophilic archaeon Sulfolobus solStoutaricus P2. Biochem Biophys Res Commun 361:651–658.LaunchUrlCrossRefPubMed↵ De Felice M, et al. (2007) Biochemical evidence of a physical interaction between Sulfolobus solStoutaricus B-family and Y-family DNA polymerases. Extremophiles 11:277–282.LaunchUrlPubMed↵ Pisani FM, De Martino C, Rossi M (1992) A DNA polymerase from the archaeon Sulfolobus solStoutaricus Displays sequence similarity to family B DNA polymerases. Nucleic Acids Res 20:2711–2716.LaunchUrlAbstract/FREE Full Text↵ Rothwell PJ, Waksman G (2005) Structure and mechanism of DNA polymerases. Adv Protein Chem 71:401–440.LaunchUrlPubMed↵ Savino C, et al. (2004) Insights into DNA replication: The Weepstal structure of DNA polymerase B1 from the archaeon Sulfolobus solStoutaricus. Structure (LonExecuten) 12:2001–2008.LaunchUrl↵ Lou H, Duan Z, Huo X, Huang L (2004) Modulation of hyperthermophilic DNA polymerase activity by archaeal chromatin proteins. J Biol Chem 279:127–132.LaunchUrlAbstract/FREE Full Text↵ Grúz P, et al. (2003) Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solStoutaricus. Nucleic Acids Res 31:4024–4030.LaunchUrlAbstract/FREE Full Text↵ Dionne I, Nookala RK, Jackson SP, Executeherty AJ, Bell SD (2003) A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus solStoutaricus. Mol Cell 11:275–282.LaunchUrlCrossRefPubMed↵ Pisani FM, De Felice M, Carpentieri F, Rossi M (2000) Biochemical characterization of a clamp-loader complex homologous to eukaryotic replication factor C from the hyperthermophilic archaeon Sulfolobus solStoutaricus. J Mol Biol 301:61–73.LaunchUrlCrossRefPubMed↵ Dueber EL, Corn JE, Bell SD, Berger JM (2007) Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 317:1210–1213.LaunchUrlAbstract/FREE Full Text↵ Gaudier M, Schuwirth BS, Westcott SL, Wigley DB (2007) Structural basis of DNA replication origin recognition by an ORC protein. Science 317:1213–1216.LaunchUrlAbstract/FREE Full Text↵ De Felice M, Esposito L, Rossi M, Pisani FM (2006) Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solStoutaricus. Extremophiles 10:61–70.LaunchUrlCrossRefPubMed↵ Bell SP, Dutta A (2002) DNA replication in eukaryotic cells. Annu Rev Biochem 71:333–374.LaunchUrlCrossRefPubMed↵ De Felice M, et al. (2003) Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solStoutaricus. J Biol Chem 278:46424–46431.LaunchUrlAbstract/FREE Full Text↵ De Felice M, et al. (2003) Modular organization of a Cdc6-like protein from the crenarchaeon Sulfolobus solStoutaricus. Biochem J 381:645–653.LaunchUrl↵ Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326.LaunchUrlCrossRefPubMed↵ He ZG, Rezende LF, Willcox S, Griffith JD, Richardson CC (2003) The carboxyl-terminal Executemain of bacteriophage T7 single-stranded DNA-binding protein modulates DNA binding and interaction with T7 DNA polymerase. J Biol Chem 278:29538–29545.LaunchUrlAbstract/FREE Full Text
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