Archaeal Eukaryote-Like Orc1/Cdc6 Initiators Physically Interact with DNA Polymerase B1 and Regulate Its Functions

Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions

Lu Zhang1, Lei Zhang1, Yi Liu1, Shifan Yang, Chunhui Gao, Hongchao Gong, Ying Feng, and Zheng-Guo He2

National Key Laboratory of Agricultural Microbiology, Center for Proteomics Research, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Edited by Charles C. Richardson, Harvard Medical School, Boston, MA, and approved April 6, 2009 (received for review December 21, 2008) Archaeal DNA replication machinery represents a core version of Based on primary structure similarities, DNA polymerases have that found in eukaryotes. However, the proteins essential for the been classified into at least 6 major families, denoted A, B, C, D, X, coordination of origin selection and the functioning of DNA poly- and Y (21). The DNA polymerases B1 from S. solfataricus is a merase have not yet been characterized in archaea, and they are member of the B family (19, 20, 22). The gene for the S. solfataricus still being investigated in eukaryotes. In the current study, the B1 DNA polymerase (SsoPolB1) encodes an 882-residue polypep- Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus tide chain with a deduced molecular mass of Ϸ100 kDa (20). The solfataricus were found to physically interact with its DNA poly- crystal structure and conserved sequence motifs of SsoPolB1 reveal merase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the a3Ј–5Ј exonuclease domain at the N terminus whereas the C DNA-binding ability of SsoPolB1 and differentially regulated both terminus coded for right-handed polymerase domains (19, 22). its polymerase and nuclease activities. Furthermore, the proteins SsoPolB1 can degrade both ssDNA and dsDNA at similar rates and also mutually regulated their interactions with SsoPolB1. In addi- specifically recognizes the presence of the deaminated bases hypo- tion, SsoPolB1c467, a nuclease domain-deleted mutant of SsoPolB1 xanthine and uracil, in a template by stalling DNA polymerization defective in DNA binding, retains the ability to physically interact 3–4 bases upstream from these lesions (23, 24). In addition, several with SsoCdc6 proteins. Its DNA polymerase activity could be archaeal replicative factors have been reported to regulate the BIOCHEMISTRY stimulated by these proteins. We report on a linkage between the activity of SsoPolB1, including proliferating cell nuclear antigen initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. (PCNA) heterotrimer (25), replication factor C (26), and the Our present and previous ﬁndings indicate that archaeal Orc1/Cdc6 conserved 7-kDa DNA-binding proteins of S. solfataricus (23). proteins could potentially play critical roles in the coordination of Both the Orc1/Cdc6 proteins and SsoPolB1 appear to be key origin selection and cell-cycle control of replication. protein components for archaeal replication (16, 23). However, the physical or functional interactions between the Orc1/Cdc6 proteins ͉ DNA replication Archaea and SsoPolB1 have not yet been characterized in any archaeal species. Three S. solfataricus Orc1/Cdc6 proteins (SsoCdc6s) are he replication of DNA is a tightly regulated process that is known to have multiple functions during the early events of DNA Tessential to all 3 domains of life: Bacteria, Eukarya, and replication (16, 18). In the present study, we have further investi- Archaea (1, 2). In the archaea, the replication machinery appears gated the potential functions of these SsoCdc6s by characterizing to be a core version of that found in eukaryotes. Therefore, archaeal the interactions between 3 SsoCdc6 proteins and SsoPolB1 from DNA replication is an excellent model system for studying the key the S. solfataricus. We report on the physical and functional links events that occur during eukaryotic DNA replication (1, 3–5). between Cdc6 proteins and SsoPolB1 in an archaeon. In contrast to the single and clearly defined sites of bacterial replication, eukaryotic DNA replication involves the ordered as- Results sembly of a number of replication factors at multiple origin sites. SsoCdc6 Proteins Physically Interact with SsoPolB1. Correlations These are bounded by a 6-subunit (Orc1–6) complex, the origin between Orc1/Cdc6 proteins and DNA polymerase have not yet recognition complex (ORC) (3, 6–10). In the archaea, DNA been characterized in any archaeal species. In this study, we used a replication proteins are found to more closely resemble the proteins bacterial 2-hybrid technique to detect the interactions between present in eukaryotes than those in bacteria (11, 12). Archaeal SsoCdc6 proteins and DNA polymerase SsoPolB1 of S. solfataricus. species also possess some of the prereplicative complex (pre-RC) As shown in Fig. S1, a positive cotransformant grew on our selective components common to eukaryotes, including a minichromosome screening medium, but the corresponding negative cotransformant maintenance (MCM)-like replicative helicase (11). In addition, they showed no growth. All 3 SsoCdc6s cotransformants could grow on have 1 or more copies of Orc1/Cdc6 proteins that show high this medium, although cotransformant strains with SsoCdc6-2 and sequence relationship to both Orc1 and Cdc6 of eukaryotes (11, 14). SsoPolB1 showed the strongest growth (Fig. S1). Therefore, we Within these archaeal homologs of eukaryotic replication proteins, were able to observe interactions between all 3 SsoCdc6s and the most likely candidates for initiator proteins are the Orc1/Cdc6 SsoPolB1. proteins, because they have been demonstrated to specifically bind To confirm the interactions between the proteins detected in the to replication origins in both in vivo and in vitro studies (14–16). In the hyperthermophilic archaeon Sulfolobus solfataricus, 3 Orc1/ Cdc6 proteins have been identified and a distinct subset of these Author contributions: Z.-G.H. designed research; Lu Zhang, Lei Zhang, Y.L., S.Y., and H.G. Cdc6 proteins is preferentially bound to each origin (16, 17). performed research; Z.-G.H. contributed new reagents/analytic tools; C.G., Y.F., and Z.-G.H. However, the Orc1/Cdc6 proteins appear to have multiple analyzed data; and Z.-G.H. wrote the paper. functions, which include both origin recognition and MCM The authors declare no conﬂict of interest. loading (16, 18). This article is a PNAS Direct Submission. In addition to the pre-RC components, the archaeal species also 1Lu Zhang, Lei Zhang, and Y.L. contributed equally to this work. contain many DNA polymerases (19, 20). DNA polymerases have 2To whom correspondence should be addressed. E-mail: [email protected]. one of the most essential and almost universal roles in the trans- This article contains supporting information online at www.pnas.org/cgi/content/full/ mission of genetic information from one generation to the next (21). 0813056106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813056106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 3, 2021 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 acti- vated by saturating the nitrilotriaceticacid sites with running buffer [100 mM Hepes⅐NaOH (pH 7.5), 50 ␮M 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-down/Western blotting assays. One hundred micrograms of SsoCdc6 protein or SsoPolY was mixed with His-tagged SsoPolB1 (100 ␮g) as described 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 marked by L, and those that eluted with buffer A containing 400 mM imidazole are marked 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 described in Materials and Methods. Coim- munoprecipitated 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. Input samples were generated as described (16). DNA recovered from the immunoprecipitates was ampliﬁed with primers speciﬁc for either the 3 oriC or a distal control region Irs14 gene.

bacterial 2-hybrid experiments, a surface plasmon resonance (SPR) SsoCdc6 proteins and SsoPolB1 were apparent, with the strongest assay was also conducted to characterize the interactions between interaction occurring with SsoCdc6-2. SsoCdc6s and SsoPolB1. A 6ϫHis-tagged PolB1 protein was im- The physiological significance of these in vitro reactions was mobilized on a nitrilotriacetate (NTA) chip. When an increasing studied with further coimmunoprecipitation (Co-IP) and ChIP amount of SsoCdc6 protein (120, 240, 480, and 960 nM) was passed experiments. An in vivo physical interaction between SsoCdc6-1 over the chip, a strong response of Ϸ1,200 response units (RU) was and SsoPolB1 was tested by using Protein A beads that were first observed for SsoCdc6-2 (Fig. 1A). A lesser, but significant, response conjugated with antibody raised against SsoPolB1 or SsoCd6-1. As could be also observed for SsoCdc6-1 (150 RU) and SsoCdc6-3 (130 shown in Fig. 1C, SsoCdc6-2 clearly associated with PolB1 as an RU) (Fig. 1A). Therefore, all 3 of the SsoCdc6 proteins interacted obvious and specific hybridization signal was detected (lane 2). with SsoPolB1 with the interaction of SsoCdc6-2 with SsoPolB1 However, no specific signal was detected for the association of being the strongest. SsoCdc6-1 with SsoCdc6-2 (Fig. 1C, lane 4), a result consistent with A pull-down/Western blotting assay was used to further charac- the previous observations using a 2-hybrid assay (17). An in vivo terize the interaction of SsoCdc6s and 6ϫHis-tagged PolB1 protein. interaction of SsoPolB1 with the other 2 SsoCdc6 proteins has not Another archaeal polymerase, PolY, previously shown to interact been detected stably. In a further ChIP assay, association of 3 with PolB1 (19), was used as a positive control. After 10% SDS/ SsoCdc6 proteins and SsoPolB1 with 2 origins, oriC2 and oriC3, PAGE and Western blotting assays using anti-SsoCdc6 or anti- were confirmed (Fig. 1D). This result is consistent with a previous SsoPolY antibody, a hybridization signal for SsoPolY (Fig. 1B, lane finding that 3 SsoCdc6 proteins bind to the oriC2 (16). Interestingly, 1) and an obvious hybridization signal for SsoCdc6-2 were seen here we found that 3 SsoCdc6 proteins bound to the oriC3. (Fig. 1B, lane 5). A weak signal was detected for SsoCdc6-1(Fig. 1B, However, the association of SsoCdc6 and PolB1 proteins with oriC1 lane 8). No signal was observed for SsoCdc6–3 (Fig. 1B, lane 11). was not stably detected. No protein was shown to bind with a distal To further confirm the findings, a 6ϫHis-tagged SsoCdc6 protein control region Irs14 gene (used as a negative control) (Fig. 1D). was used to characterize physical interactions between PolB1 and SsoCdc6 proteins (Fig. S2). The 3 SsoCdc6 proteins and a positive SsoCdc6 Proteins Stimulate the Binding of SsoPolB1 to the Template/ control, SsoPCNA2 (16), were observed to interact with SsoPolB1. Primer DNA Substrate. Previous studies have shown that both BSA did not interact with either SsoCdc6 or SsoPolB1 proteins SsoCdc6 protein and SsoPolB1 can bind to the replication fork-like (Fig. S2). Differences in the strength of the interactions between DNA substrate (23). This finding suggested that the physical

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813056106 Zhang et al. Downloaded by guest on October 3, 2021 same DNA substrate as above (Fig. 2A). When 0.9 ␮M SsoPolB1 and 200 fmol of 32P-labeled DNA were mixed together 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 contrast, 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. We then examined the nuclease activity of SsoPolB1 by using the same DNA as a substrate. When 0.9 ␮M SsoPolB1 and 200 fmol of 32P-labeled DNA were mixed together, 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 effect 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 Fig. 2. Effects of SsoCdc6s on the DNA-binding activity of SsoPolB1 onto the the DNA polymerase and the nuclease activities of SsoPolB1. template/primer DNA substrate An EMSA was used to assess the effect of the However, differences existed in the overall effectiveness of each SsoCdc6 proteins on the DNA-binding activities of the SsoPolB1. The experiments were carried out as described in Materials and Methods.(A) The type of SsoCdc6 protein. structures and sequences of the DNA substrate used in EMSAs. The DNA BIOCHEMISTRY substrate was produced by annealing a 34-bp Oligo and a 76-bp Oligo, thus it SsoCdc6 Proteins Mutually Regulate Their Interactions with SsoPolB1. contains a partial double strand and leaves a single-strand tail. (B) The EMSAs When the SsoPolB1 polymerase activity was measured on the DNA were carried out to detect the DNA-binding abilities of SsoCdc6-1 (1.8 ␮M), substrate in the presence of different pairs of SsoCdc6 proteins, SsoCdc6-2 (1.8 ␮M), SsoCdc6-3 (1.8 ␮M), and SsoPolB1 (0.9 ␮M), or the mutual regulations of SsoPolB1 by the protein pairs could be SsoPolB1 in combination with different SsoCdc6 proteins. Speciﬁc protein/ characterized. When SsoCdc6-2 levels were kept constant, a step- DNA complex is indicated. wise increase in inhibition was observed with stepwise addition of either SsoCdc6-1 or SsoCdc6-3 (Fig. 3A, lanes 13–18). In contrast, with a fixed amount of SsoCdc6-1, a stepwise decrease in inhibition interactions between the 2 proteins might exist that could modulate was seen with stepwise increases in SsoCdc6-3 (Fig. 3A, lanes the DNA-binding ability of SsoPolB1. We therefore conducted an 19–21). EMSA using a template/primer DNA as the substrate (Fig. 2A) With a fixed concentration of SsoCdc6-2 in the reaction mixture, (23). The DNA substrate contained a partial double strand that left the inhibition of nuclease activity of SsoPolB1 became somewhat a single-strand tail (Fig. 2A). As shown in Fig. 2B, a specific stronger with stepwise increases in the amount of SsoCdc6-1 (Fig. protein/DNA complex was observed for each of the SsoCdc6 ␮ 3B, lanes 12–14). In contrast, the inhibition became much weaker proteins or SsoPolB1 (0.3 M) alone. When a different SsoCdc6 when an increasing amount of SsoCdc6-3 was added (Fig. 3B, lanes was mixed with SsoPolB1, no specific SsoCdc6/DNA complex was 15–17). With a fixed amount of SsoCdc6-1, the inhibition declined observed under these conditions. Thus, SsoPolB1 inhibited the to almost 0 as the amount of SsoCdc6-3 was increased (Fig. 3B, binding of SsoCdc6 onto the DNA substrate (Fig. 2B, lanes 6–8). lanes 18–20). It is likely that the physical interaction between the SsoPolB1 and These results indicated that SsoCdc6 proteins could mutually SsoCdc6 proteins negatively affected their ability to bind DNA. In regulate their effects on both polymerase and nuclease activity of contrast, SsoCdc6 proteins stimulated the binding of SsoPolB1 to SsoPolB1. In particular, partial recovery of the inhibition imposed the DNA substrate, because an obviously stronger SsoPolB1/DNA by SsoCdc6-1 was achieved by the addition of SsoCdc6-3 for both complex band was observed on the gel (Fig. 2B, lanes 6–8). In the polymerase and nuclease activities of SsoPolB1. These func- particular, a slower-running DNA/protein complex band was ob- tional effects suggest a mutual regulation by the SsoCdc6 proteins served when SsoCdc6–2 was mixed with SsoPolB1 (Fig. 2B, lane 6). in their physical interactions with SsoPolB1. This idea was further This band most likely represented the SsoCdc6–2/SsoPolB1/DNA investigated by SPR assay. A result similar to that shown in Fig. 1A complex because it has a larger size than does the SsoPolB1/DNA was observed for the interaction of the single SsoCdc6 with complex (Fig. 2B, lanes 2, 7, and 8). Larger complexes were also SsoPolB1 (Fig. 3C Left). When compared with the response gen- observed when SsoPolB1 was combined with a mixture of 2 erated by interaction of the SsoCdc6-2 alone with SsoPolB1 SsoCdc6 proteins, either SsoCdc6-2/SsoCdc6-1 or SsoCdc6-2/ (Ϸ2,000 RU), addition of either SsoCdc6-1 or SsoCdc6-3 together SsoCdc6-3 (Fig. 2B, lanes 9 and 10). No larger complex was with SsoCdc6--2 substantially reduced the response signal (Ϸ750 observed for SsoCdc6-1/SsoCdc6-3 (Fig. 2B, lane 11). 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 Both the DNA Polymerase and the Nuclease Activities of SsoPolB1. In the above studies, we found that SsoCdc6 SsoCdc6 Proteins Regulate the Function of SsoPolB1c467. The proteins physically interacted with SsoPolB1 and stimulated its SsoPolB1 of S. solfataricus contains DNA polymerase and nuclease DNA-binding activity. To examine the functional linkage between domains (23). We cloned and purified SsoPolB1c467, the C- these SsoCdc6 proteins and SsoPolB1, we analyzed the regulatory terminal DNA polymerase domain of SsoPolB1 (Fig. 4A). Using an effects of SsoCdc6 proteins on the activities of SsoPolB1. EMSA, SsoPolB1c467 showed negligible DNA-binding activity The polymerase activity of SsoPolB1 was measured by using the when compared with the wild-type SsoPolB1 (Fig. 4B). However,

Zhang et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 3, 2021 A SsoPolB1 1

B SsoCdc6-2 SsoCdc6-1 lo A A Po SsoCdc6-2SsoCdc6-1 SsoCdc6-3 SsoCdc6-1 SsoCdc6-3 SsoCdc6-3 N ND DNA substrate s D S 5’ * 3’ 42bp lanes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 3’ 5’ 76bp

DNA polymerase products

42bp DNA substrate

SsoPolB1 1 B

B loPo SsoCdc6-2 SsoCdc6-1 A s ND SsoCdc6-2 SsoCdc6-1 SsoCdc6-3 SsoCdc6-1 SsoCdc6-3 SsoCdc6-3 S DNA substrate 5’ * 3’ 42bp lanes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3’ 5’ 76bp

DNA substrate

DNA exonuclease products

C SsoPolB1/ SsoCdc6 SsoPolB1/ a pair of SsoCdc6s

Fig. 3. Effects 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 described 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 ␮M) in the presence of a fixed amount of SsoPolB1 (0.9 ␮M) alone or in combination with another 0.9 ␮M 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 ␮M) in the presence of a fixed amount of SsoPolB1 (0.9 ␮M) alone or in combination with another 0.9 ␮M SsoCdc6 protein. (C) SPR assays for the mutual effects of SsoCdc6 proteins on the interactions with SsoPolB1. The experiments were carried out as described in Materials and Methods. Each analysis was performed in triplicate. An overlay plot was produced for depicting the interactions between 1 ␮M SsoCdc6 alone and SsoPolB1 (Left) or between a pair of SsoCdc6 proteins with SsoPolB1 (Right). Representative data are shown. 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.

in the bacterial 2-hybrid assay (Fig. S1A), all 3 SsoCdc6 proteins Discussion retained their ability to interact with SsoPolB1c467. The interac- Effective DNA replication requires coordinated and tightly- tions of both SsoCdc6-2 and SsoCdc6-3 with SsoPolB1c467 ap- regulated protein–protein and protein–DNA interactions. How- peared to be even stronger than those with the wild-type SsoPolB1. ever, in archaeal species, the physical or functional interactions As shown in Fig. 4C, SsoPolB1c467 only retained a very weak DNA between the Orc1/Cdc6 proteins and DNA polymerase have not yet polymerase activity (lane 3) compared with the wild-type protein been characterized. In the present study, we have characterized the (lane 1). However, the activity of SsoPolB1c467 could be stimulated physical and functional interactions of 3 SsoCdc6 proteins with the by all 3 of the SsoCdc6 proteins, although to a different extents. DNA polymerase, SsoPolB1, of S. solfataricus. We found that these SsoCdc6–3 caused the greatest stimulation (Fig. 4C, lanes 8 and 9). SsoCdc6 proteins regulate both the DNA polymerase and the Therefore, because of its retention of physical interaction with nuclease activities of SsoPolB1 and this regulation can occur SsoCdc6 proteins, the DNA polymerase activity of SsoPolB1c467 through mutual interactions of these 3 proteins. could be stimulated by these proteins in the absence of DNA- The interactions between SsoCdc6 proteins might facilitate the binding ability. assembly of a cooperative complex at the origin, based on the

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813056106 Zhang et al. Downloaded by guest on October 3, 2021 appears to be unique because it interacts clearly with SsoMCM and A Exonuclease domain Polymerase domain SsoPolB1, but also with other SsoCdc6 proteins (18, 27, 28). A SsoPolB1 similar protein in eukaryotes is Cdc45, which associates with DNA polymerase, ORC, and the MCM proteins (30). These associations SsoPolB1c467 have been suggested to indicate that Cdc45 coordinates the func-

76 tioning of these components in the replication fork (30). To date,

B 4c1 a homolog of the eukaryotic Cdc45 has not yet been characterized 1BloPosS

BloP in any of the genome-sequenced archaeal species. From the current A o

ND study, SsoCdc6-2 would appear to play an analogous role to that of sS DNA substrate lanes 1 2 3 Cdc45. Therefore, like Cdc45 in eukaryotes, it may play a pivotal 5’ * 3’ 42bp role in the transition of DNA replication from initiation to exten- 3’ 5’ 76bp sion. 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 DNA polymerase/DNA situate it to the origin, after which the archaeal MCM and poly- complex merase 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. DNA substrate SsoPolB1 has been reported to specifically recognize the presence of deaminated bases in a template by stalling DNA synthesis C 764c1BloPosS SsoPolB1c467 3–4 bases upstream of these lesions (24). Consequently, SsoPolB1 1Blo has been proposed to function in DNA repair processes (22). In the A

PosS SsoCdc6-2 SsoCdc6-1 SsoCdc6-3 ND current study, we present evidence that the SsoCdc6 proteins can regulate both the DNA polymerase and nuclease activities of lanes 123456789 SsoPolB1. We found that the 3 SsoCdc6 proteins also mutually BIOCHEMISTRY 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 meta- DNA substrate bolic processes such as DNA repair and DNA lesion reactions. It would be interesting to investigate the interaction between archaeal 5’ * 3’ 42bp 3’ 5’ 76bp 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, show that archaeal Orc1/Cdc6 proteins have multiple functions, Fig. 4. Effects of SsoCdc6s on the activity of SsoPolB1 DNA polymerase including origin recognition, MCM loading, DNA polymerase domain. (A) Schematic representations of the polypeptide chain of SsoPolB1 switching, and even DNA repair. These results raises the interesting proteins and its N-terminal-deleted mutant protein (SsoPolB1c467) (23). (B) possibility that the SsoCdc6-2 protein itself is a core regulator of The EMSA was used to compare the DNA-binding activities of both SsoPolB1 (0.9 ␮M) and SsoPolB1c467 (0.9 ␮M). The experiments were carried out as archaeal DNA replication and that it may facilitate an integrated described in Materials and Methods.(C) The effects of SsoCdc6 proteins on process for coordination between origin selection and cell cycle DNA polymerase activities of SsoPolB1c467 were carried out with increasing control of replication. The ability to study these events in archaea amounts of SsoCdc6 (1.35 and 1.8 ␮M) in the presence of a fixed amount of could prove to be a powerful tool for addressing related mechanistic SsoPolB1c467 (0.9 ␮M) by using the same template/primer DNA substrate. The issues in eukaryotic cell-cycle control of replication. experiments were carried out as described in Materials and Methods. Specific products of the enzyme are indicated on the right. Materials and Methods DNA and Oligonucleotides. Oligonucleotides were synthesized by Invitrogen. The template/primer DNA substrates (Fig. 2A) were constructed by annealing the existence of binding sites for the 3 SsoCdc6 proteins on the origin labeled p42 (5Ј-CAGTGAATTCGAGCTCG GTACCCGGGGATCCTCTAGAGTCGA-3Ј) (16, 18, 27–29). However, differential interactions of the 3 SsoCdc6 at the 5Ј end of the primer, with a 3-fold molar excess of the cold complementary proteins with SsoMCM have also been observed (18). SsoCdc6-2 strands t76 (3Ј-TTTTTGTCACTTAAGCTCGAGCCATGGGCCCCTAGGAGATCT CA stimulates the binding of the SsoMCM to the origin DNA, whereas GCTGGACGTCCGTACGTTCGAACCGCATTTTT-5Ј) (23). SsoCdc6-1 and SsoCdc6-3 significantly inhibit this activity (18). In the current study, we found that the 3 SsoCdc6 proteins also have Cloning and Purification of SsoCdc6 and SsoPolB1 Proteins. Prokaryotic vectors significant roles in the coordination of the DNA polymerase activity expressing the genes for SsoCdc6 and SsoPolB1 proteins and untagged proteins of S. solfataricus SsoPolB1. These roles are likely to be different, were constructed as described (18) (Table S1). Escherichia coli BL21 CodonPlus based on the different degrees of interaction observed with the (DE3)-RIL cells (Novagen) were used as the host strain to express archaeal proteins DNA polymerase. In particular, SsoCdc6-2 had the strongest as described (18, 23, 31, 32). Protein concentrations were determined by spectro- interaction with SsoSsoPolB1, which may imply that SsoCdc6-2 photometric absorbance at 280 nm according to Gill and Hippel (33). functions might not be limited only to early replication events such Bacterial 2-Hybrid Analysis. Bacterial 2-hybrid analysis was carried out according as origin recognition and MCM loading. Rather, this protein may to the procedure supplied with the commercial kit. pBT and pTRG vectors con- also be involved in the coordination of the function of the DNA taining archaeal genes of SsoCdc6 and SsoPolB1 were generated. All of the polymerase. primers used for PCR amplification are described in Table S2. Positive growth Previous reports, and the current study, have indicated that all 3 cotransformants were selected on our selective screening medium plate contain- of the SsoCdc6 proteins can interact with the replication origin, ing 5 mM 3-amino-1,2,4-triazole (3-AT) (Stratagene), 8 ␮g/mL streptomycin, 15 SsoMCM, and SsoPolB1 (16, 18). Of the 3 proteins, SsoCdc6-2 ␮g/mL tetracycline, 34 ␮g/mL chloramphenicol, and 50 ␮g/mL kanamycin.

Zhang et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on October 3, 2021 Pull-Down and Western Blot Assays. Pull-down assays were carried out as EMSAs. The binding of SsoCdc6 or SsoPolB1 proteins to DNA was performed on described (18). One hundred micrograms of SsoCdc6 protein or SsoPolY was a template/primer DNA by using a modiﬁed EMSA as described (18, 34). The mixed with His-tagged SsoPolB1 (100 ␮g) into 500 ␮L of incubation buffer reactions (10 ␮L) for measuring the mobility shift contained 200 fmol of 32P-

containing 20 mM Tris⅐HCl (pH 7.5), 100 mM NaCl, and 0.5 mM MgCl2 and labeled duplex DNA and various indicated amounts of SsoCdc6 proteins concen- incubated at 25 °C for 10 min, then coincubated at 4 °C for 1 h with Ni-NTA trations diluted in buffer containing 20 mM Tris⅐HCl (pH 7.5), 100 mM NaCl, 2 mM agarose. The beads were then washed 2 times with 1 mL of buffer containing 20 EDTA, 0.5 mM MgCl2, and 0.7 mM 2-mercaptoethanol. The gel was dried and mM imidazole and centrifuged at 800 ϫ g for 1 min. Proteins bound to the beads analyzed by using a modification of published procedures (18). were eluted with 100 ␮L of elution buffer A containing 400 mM imidazole. The eluates were then analyzed by 10% SDS/PAGE and Western blot using anti- Nuclease Activity Assays. The nuclease activity analysis was performed on the SsoCdc6 or anti-SsoPolY antibodies. template/primer DNA by using a modification of published procedures (23). The reactions (10 ␮L) for measuring the activity contained 200 fmol of 32P- labeled partial duplex DNA and various indicated amounts of SsoCdc6 pro- Co-IP and ChIP Assays. The in vivo interactions between SsoCdc6 proteins teins concentrations diluted in buffer containing 10 mM Tris⅐HCl (pH 7.5), 1 and SsoPolB1 were analyzed by Co-IP and ChIP. Exponentially growing cells mM DTT, 100 ␮g/mL BSA, and 10 mMMgCl . The samples were analyzed by of S. solfataricus were harvested, resuspended, and lysed in 4 mL of buffer 2 electrophoresis in a 16% polyacrylamide (acr/bis 19:1), 8 M urea gel in 0.5ϫ Tris [50 mM Tris⅐HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40]. borate–EDTA (TBE). The gel was dried and analyzed by using a modification of ␮ Co-IPs were performed by incubating 10 g of archaeal cell extract with 3 published procedures (23). ␮L of SsoPolB1 (or SsoCdc6) antiserum in 100 ␮L of buffer for3hat4°Cwith shaking. A 20-␮L slurry of protein A Sepharose was added, and incubation DNA Polymerase Activity Assays. DNA polymerase activity was performed on the was continued for another hour. Immune complexes were collected, and template/primer DNA by using a modification of published procedures (23). The the beads were washed with buffer. Finally, the beads were resuspended reactions (10 ␮L) contained 200 fmol of 32P-labeled duplex DNA and various in SDS/PAGE sample buffer. After boiling, the samples were analyzed by indicated amounts of SsoCdc6 proteins concentrations diluted in buffer contain- Western blotting using anti-SsoCdc6 (or anti-PolB1) antibody. Interactions ing 20 mM Tris⅐HCl (pH 7.5), 100 mM NaCl, 2 mM EDTA, 0.5 mM MgCl2, 0.7 mM of the 3 SsoCdc6 proteins with SsoPolB1 at the origins were analyzed by 2-mercaptoethanol, and 40 mM dNTPs. The reaction was incubated for 20 min at using the ChIP technique as described (16). ChIP used preimmune or 65 °C, unless otherwise specified, and quenched by the addition of 10 ␮Lof immune sera raised against SsoCdc6 or SsoPolB1. DNA recovered from ice-cold 2ϫ loading buffer (95% deionized formamide, 100 mM EDTA, 0.02% immunoprecipitates was amplified with primers specific for 3 oriC or a bromophenol blue) (23). Reactions were incubated at room temperature for 20 distal control region Irs14 gene (16). min before loading onto 5% polyacrylamide/bis (37.5:1) gels. The gel was dried and analyzed by using a modification of published procedures (23). SPR. Physical interactions of SsoCdc6 proteins with SsoPolB1 were analyzed on a BIAcore 3000 instrument (GE Healthcare) as described (34). The SsoPolB1protein ACKNOWLEDGMENTS. We thank Prof. Charles C. Richardson for constructive was immobilized on NTA chips. The purified SsoCdc6 proteins to be used as the suggestions and Prof. Huang Li (Institute of Microbiology, Chinese Academy of Sciences) for archaeal strains. This work was supported by the National Natural ligands were then diluted in running buffer [10 mM Hepes (pH 7.4), 150 mM NaCl, Science Foundation of China, 973 Program Grant 2006CB504402, New Century ␮ 50 M EDTA, 0.005% BIAcore surfactant P20]. Each analysis was performed in Excellent Talents Fund of the Ministry of Education of China Grant NECT-06-0664, triplicate. An overlay plot was produced to distinguish the interactions between Doctoral Fund of Ministry of Education of China Grant 200805040004, and China replication proteins. Representative data are shown in the figures. National Fundamental Fund of Personnel Training Grant J0730649.

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