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1 Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
2 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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S-Cdk is required during the S phase to phosphorylate Sld2p and other protein substrates (Masumoto et al. 2002). Dbf4p, whose expression is cell-cycle dependent, forms a heterodimer with and regulates the kinase activity of Cdc7p (Johnston et al. 1999; Masai & Arai 2002). Ddk phosphorylates Mcm2p and other replication proteins in budding yeast and other eukaryotic cells and is required for activation of early- and late-firing origins throughout the S phase in S. cerevisiae (reviewed in Masai & Arai 2002). It has been suggested that the Mcm4/6/7 heterohexamer, which possesses DNA helicase activity in vitro (Ishimi 1997), is one of the key replicative DNA helicases, because Mcm2-7p translocates along DNA with the replication fork during the S phase and because Mcm2-7p is required for completion of DNA replication (Aparicio et al. 1997; Labib et al. 2000). However, there is no direct evidence that Mcm4/6/7 is active as a DNA helicase at the replication fork.
In S. cerevisiae, DNA sequences that act as replicator elements, origins of bidirectional replication and many key replicative enzymes proteins have been characterized. Replication initiation has been observed in vitro using nuclei from G1-arrested cells and S phase protein extracts (Pasero et al. 1997; Mitkova et al. 2005). However, because Cdk has both positive and negative effects on replication, it might not be possible to assemble pre-RC and activate replication in the same extracts. To solve this problem, Seki & Diffley (2000) developed an in vitro system in which pre-RC is assembled using extracts from G1-arrested cells, origin DNA bound to magnetic beads, ATP and Cdc6p. Recently, sequential ATP hydrolysis by Cdc6p and ORC was shown to be critical for the loading of Mcm2-7p in vitro (Bowers et al. 2004; Randell et al. 2006), mirroring the requirements for pre-RC assembly in vivo. However, in vitro pre-RC assembly is inefficient, probably due to use of a linear DNA substrate and/or insufficient quantities of pre-RC components in extracts from G1-arrested cells.
The goal of this study was to develop an efficient in vitro system for pre-RC assembly. To this end, highly concentrated protein extracts were used to initiate DNA replication on a plasmid DNA substrate carrying 20 tandem copies of ARS1. The results revealed sequential recruitment of ORC, Cdc6p, and Cdt1p-Mcm2-7p on to ARS1. When Mcm2-7p was maximally loaded, Cdc6p and Cdt1p were released with similar kinetics. Furthermore, Cdt1p-Mcm2-7p from G1-arrested, early S or G2/M-arrested cells exhibited structure-specific DNA binding and was loaded on to ORC-Cdc6p during pre-RC assembly. Finally, using highly purified ORC, Cdc6p and Cdt1p-Mcm2-7p, pre-RC assembly was fully reconstituted in vitro in the presence of ATP and ARS1.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Previous studies demonstrated in vitro assembly of the pre-RC on a yeast replication origin in a reaction that requires ATP, Cdc6p and extracts from G1-arrested yeast cells (Seki & Diffley 2000). We modified this pre-RC assembly assay in two ways to increase its efficiency and facilitate analysis of molecular dynamics during pre-RC assembly. First, yeast cells were disrupted with an electric mortar in the presence of liquid nitrogen, which yielded highly concentrated protein extracts. Second, a plasmid DNA substrate that carries 20 tandem copies of ARS1 was used, since multiple origins of replication suppress mitotic loss in cdc6 and cdc14 mutant cells (Hogan & Koshland 1992). The efficiency of pre-RC assembly was ARS1-specific and roughly proportional to ARS1 copy number (data not shown and Fig. 1A).
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A hallmark of pre-RC formed in vivo is that, once assembled on to origin DNA, Mcm2-7p is resistant to salt extraction. In contrast, ORC and Cdc6p can be extracted from chromatin using high-salt washes (Rowles et al. 1996; Donovan et al. 1997; Edwards et al. 2002). Similar results were observed in vitro. Thus, ORC, Cdc6p and Cdt1p were readily dissociated by high salt from pre-RC formed in vitro, but Mcm2p (one of the representative subunit of Mcm2-7p) was only partially dissociated by similar treatment (Fig. 1D). Because Mcm2-7p does not bind origin DNA directly in the absence of ORC and Cdc6p, these data suggest that pre-RC reconstitution in vitro reflects a true pre-RC assembly reaction, and is not simply the result of physical interactions between origin-bound ORC and other replicative proteins.
Purified ORC and rCdc6p load Cdt1p and Mcm2-7p on to ARS1
Because Cdc6p is present at low levels even in 
-factor-arrested cells (Piatti et al. 1995; Drury et al. 1997), we used protein extracts from G1-arrested yeast cells over-expressing Cdc6p as published (Seki & Diffley 2000). Because soluble ORC is also present at a very low level in G1-arrested cells, purified ORC and recombinant Cdc6p (rCdc6p) were added to protein extracts prior to pre-RC assembly (Fig. 2A). Figure 2B shows that binding of ORC to ARS1 increased two- to threefold and binding of Cdc6p, Mcm2-7p and Cdt1p increased up to tenfold as the amount of input ORC increased. Addition of rCdc6p to Cdc6p-depleted G1-arrested cell extracts stimulated binding of Mcm2-7p and Cdt1p to ARS1, but did not stimulate binding of ORC (Fig. 2C). Lower efficiency of binding was achieved with extracts from Cdc6p-over-expressing cells than with exogenously added rCdc6p, possibly due to partial inactivation of Cdc6p in extracts of G1-arrested cells. Previous studies show that Cdc6p is stabilized when it is over-expressed in cdc4-1 cells (Drury et al. 1997), and extracts from these cells also loaded Cdc6p and Mcm2-7p more efficiently than extracts from wild-type cells (data not shown).
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Previous attempts to purify Mcm2-7p from S. cerevisiae have not succeeded, although Mcm2-7p has been purified from Schizosaccharomyces pombe (Adachi et al. 1997). In this study, tandem affinity purification (TAP) (Puig et al. 2001) followed by glycerol density gradient centrifugation was used to purify S. cerevisiae Mcm2-7p. When Mcm2-7p was purified from G1- or G2/M-arrested cells, the complex copurified with approximately equimolar Cdt1p (Fig. 3C); when purified from early S phase cells, Mcm2-7p was isolated in one of two forms: form S1, with a trace amount of Cdt1p or form S2, with a significant amount of Cdt1p (Fig. 3C).
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Loading of purified Cdt1p and Cdt1p-Mcm2-7p as a part of pre-RC in vitro
The role of Cdt1p in pre-RC assembly was investigated using sid2-21 cells (Jacobson et al. 2001; Devault et al. 2002), which lack detectable Cdt1p but express a normal level of Mcm2p (data not shown). sid2-21 cells over-expressing Cdc6p were grown at 30 °C, arrested in G1 with -factor, and extracts from these cells were used in pre-RC assembly reactions. During pre-RC assembly with sid2-21 extracts, ORC and Cdc6p load on to ARS1 with normal kinetics and Cdc6p dissociates from the complex after 16 min (Fig. 4A). However, neither Cdt1p nor Mcm2p load on to ARS1 in the mutant extracts at 24 °C, suggesting that Cdt1p is required for the loading of Mcm2-7p on to ARS1. When purified Cdt1p was added to the reaction, Mcm2-7p loaded on to ARS1 normally at 24 °C suggesting that Cdt1p complements deficiency of sid2-21 mutant cells (Fig. 4B). Furthermore, when the ORC-Cdc6-ARS1 complex was isolated from an in vitro assembly reaction with sid2-21 extracts and combined with exogenous purified Cdt1p-Mcm2-7p, Mcm2p was loaded on to ARS1 normally (Fig. 4C). When purified, Cdt1p-Mcm2-7p was added directly to extracts from sid2-21 mutant cells, both Cdt1p and Mcm2p bound to ARS1 in a Cdc6p-dependent manner (data not shown). Thus, these data strongly suggest a two-step mechanism by which Mcm2-7p is loaded on to ARS1: in the first step, Cdt1p-Mcm2-7p binds to ARS1 containing ORC and Cdc6p and in the second step, Cdt1p dissociates from ARS1.
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The above experiments suggest that the minimum requirements for in vitro pre-RC assembly are ORC, Cdc6p, Cdt1p-Mcm2-7p, ATP and ARS1 DNA. This was tested by reconstituting pre-RC assembly in vitro with purified ORC, rCdc6p, and Cdt1p-Mcm2-7p. The results demonstrate efficient pre-RC assembly with these proteins in the presence of ATP and ARS1 DNA (Fig. 5A). The efficiency of pre-RC assembly was similar using Cdt1p-Mcm2-7p purified from G1-, early S (S2)- or G2/M-arrested cells (data not shown) or with extracts from cells over-expressing Cdc6p. Omission of any one of these purified proteins or ATP blocked in vitro assembly of the pre-RC (Fig. 5B,C) and addition of apyrase to deplete endogenous ATP also reduced the efficiency of pre-RC assembly (Fig. 5C). The Mcm2-7p in the product of this reaction was resistant to high salt washing, as observed for pre-RC formed using protein extracts (Fig. 5B). These results suggest that the reconstituted pre-RC is very similar to pre-RC formed with protein extracts in vitro. The results also demonstrate that Noc3p (Zhang et al. 2002) is not essential for in vitro pre-RC assembly on chromatin-free ARS1 DNA. Reconstitution of pre-RC assembly with purified ORC, rCdc6p, Cdt1p, and Mcm2-7p was also tested (Fig. 5D). It should be noted that purified Cdt1p has an affinity to the magnetic beads we used even in the absence of DNA (data not shown). Regardless of this problem, the amount of Mcm2p loaded on to ARS1 in the presence of Cdt1p was comparable to that of Mcm2p in the absence of Cdt1p (Fig. 5D), suggesting that Cdt1p cannot efficiently support the loading of Mcm2-7p in this reaction.
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Discussion |
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A method for in vitro assembly of yeast pre-RC was first developed by Seki & Diffley (2000). Here, the efficiency of that method was improved by increasing ARS1 copy number from one to twenty. Improved efficiency of pre-RC assembly was also achieved by over-expressing ORC (Bowers et al. 2004). In our system, the efficiency of pre-RC assembly increased approximately tenfold and was roughly proportional to an ARS1 copy number. The Mcm2-7p in the pre-RC formed in this system was resistant to high salt (Figs 1D and 5B), indicating that pre-RC formed in vitro has similar salt sensitivity as pre-RC formed in vivo. In addition, Mcm proteins in pre-RC were phosphorylated in a similar manner by recombinant Cdc7-Dbf4 using pre-RC assembled in vitro or in vivo (unpublished observations). These results suggest that yeast pre-RC assembled in vitro has similar properties to yeast pre-RC assembled in vivo, validating the reconstitution system described here.
Tanaka & Diffley (2002) previously showed that, like Mcm2-7p, S. cerevisiae Cdt1p accumulates in the nucleus during G1 phase and is excluded from the nucleus later in the cell cycle by Cdks. Furthermore, they showed that Cdt1p interacts with Mcm2-7p, and the nuclear accumulation of these proteins during G1 is interdependent. Consistent with those findings, we were able to purify Cdt1p-Mcm2-7p from G1-, early S-phase and G2/M-arrested cell extracts and showed that they all can be loaded on to ORC-Cdc6p bound ARS1, indicating that they are active and function as a complex during pre-RC assembly unlike other systems (reviewed in Stillman 2005). Since we extracted a majority of soluble Mcm2-7p and part of chromatin-bound Mcm2-7p with 0.5 M potassium glutamate (equivalent to about 0.2–0.3 M KCl; unpublished observations), these results also suggests that Mcm2-7p complex associates with Cdt1p, before it binds to the origin-competent chromatin during pre-RC assembly. Consistent with this proposal, we found that the total amount of each Mcm2-7p is almost the same as that of Cdt1p inside the cell (data not shown), unlike the previous measurement of each Mcm protein in yeast (Lei et al. 1996).
Cdc6p binds directly to ORC (potentially via Orc1p) and enhances the DNA binding specificity of ORC (Wang et al. 1999; Mizushima et al. 2000). Furthermore, the interaction between Cdc6p and ORC induces significant structural change in ORC (Mizushima et al. 2000). Thus, it is possible that Cdc6p, an AAA+ protein, may bind to another AAA+ protein subunit in ORC (such as Orc1p), as observed in DNA polymerase clamp loader complexes (Speck et al. 2005). The potential similarity between Cdc6p and the clamp loaders has been discussed previously, since Cdc6p is required for loading Mcm2-7p on to chromatin in vivo and in vitro (Donovan et al. 1997; Perkins & Diffley 1998; Weinreich et al. 1999; Seki & Diffley 2000). This study shows that ATP and ORC-Cdc6p bound ARS1 DNA is required for loading Cdt1p-Mcm2-7p on to ARS1 (Fig. 5). Thus, although Cdc6p may be the functional equivalent of the RF-C clamp loader, it is more likely that ORC-Cdc6p loads Cdt1p-Mcm2-7p. This reaction would be analogous to RFC loading PCNA, which recruits Cdt1p-Mcm2-7p. However, this mechanism does not explain how Cdt1p-Mcm2-7p becomes salt-resistant during pre-RC assembly.
A novel finding of this study is that Cdt1p-Mcm2-7p, but not Mcm2-7p, binds specifically to bubble- and Y-shaped DNA in the absence of ATP, while it did not bind to 3'-tailed, 5'-tailed, single-stranded, or double-stranded DNA (Fig. 3D). The biological significance of this observation and its relevance to initiation of yeast DNA replication is not known. However, it is conceivable that Cdt1p-Mcm2-7p binds to locally unwound regions of ARS1 DNA, although localized melting of ARS1 was not detected during in vitro assembly of pre-RC (data not shown). You et al. (2003) found that the helicase and ATPase activities of the mammalian Mcm4/6/7 subcomplex are activated specifically by thymine stretches. They also showed that the Mcm4/6/7 helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively. Thus, the binding specificity of Mcm4/6/7 resembles the binding specificity reported here for Cdt1p-Mcm2-7p; however, Cdt1p-Mcm2-7p lacks ATPase and DNA helicase activity and demonstrates ATP-independent structure-specific DNA binding.
The major achievement of this study is the reconstitution of a pre-RC assembly with purified ORC, rCdc6p and Cdt1p-Mcm2-7p in the presence of ATP and ARS1. This result establishes the minimum requirements for in vitro pre-RC assembly. However, it remains possible that additional factors participate in or stimulate pre-RC assembly in vivo. For example, Zhang et al. (2002) report that Noc3p interacts with Mcm2-7p and ORC and that it binds to replication origins throughout the cell cycle, although we could not observe specific binding of Noc3p on to ARS1 in vitro upon pre-RC assembly (unpublished observations). A Xenopus system for in vitro reconstitution of licensed replication origins using purified proteins and Xenopus sperm chromatin has also been reported (Gillespie et al. 2001). The protein requirements in this Xenopus system include Nucleoplasmin, XORC, XCdc6p, RLF-B/Cdt1p and XMcm2-7p but not XNoc3p (Gillespie et al. 2001). Gillespie et al. (2001) confirmed successful reconstitution by showing that the reconstituted origins support DNA replication in vitro in the presence of Xenopus egg extract and 6-DMAP, which inhibits assembly of pre-RC. However, we failed to reconstitute a pre-RC assembly with purified ORC, rCdc6p, Cdt1p and Mcm2-7p (Fig. 5D), although purified Cdt1p can support pre-RC assembly in sid2-21 extract. These results suggest that pre-assembly of Cdt1p-Mcm2-7p may be important for pre-RC assembly in S. cerevisiae.
Previous in vivo studies indicate that pre-RC assembly is associated with a change in the DNase I footprint of ORC on ARS1 (Diffley et al. 1994). Thus, the DNase I footprint of ORC could be used as a measure of proper pre-RC assembly. When the DNase I footprint of ORC was examined here using in vitro-assembled intermediates of pre-RC and a DNA substrate carrying a single copy of ARS1, the ORC footprint did not appear to change significantly during assembly (data not shown). This may indicate insufficient loading of Cdc6p or Cdt1p-Mcm2-7p (i.e. overall efficiency of pre-RC formation was about 2.5–5%; Fig. 5A). Alternatively, it may also indicate that rCdc6p is not fully active, even though it greatly stimulated binding of Cdc6p-Cdt1p-Mcm2-7p to ARS1 (Fig. 2C). Thus, although the pre-RC assembled in vitro may be structurally similar to pre-RC in vivo, additional factor(s) may be required to induce the native in vivo conformation of ORC in the pre-RC.
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Experimental procedures |
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The yeast S. cerevisiae strains used in this study are listed in Table 1. For construction of wild-type and mutant multicopy ARS1 plasmids, 173 bp ARS1 fragment which contains A (or ACS), B1, B2, and B3 elements was amplified by PCR (primers 5'-AAAATAGCAAATTTCGTC-3' and 5'-AAAGCCAAATGATTTAGC-3' were used) using pARS1/WTA or pARS1/858-865 (Marahrens & Stillman 1992) as a template and five direct repeat was constructed between KpnI and SacI sites of pBluescript KS(+). For constructing the 20 repeat of wild-type ARS1, XhoI site was introduced at KpnI site and SalI and NotI sites were introduced at SacI site. Using a combination of XhoI, SalI and NotI restriction enzymes, a 5 repeat was doubled to 10 and a 10 repeat was doubled to 20, as previously described (Robinett et al. 1996). For constructing the 20 repeat of mutant ars1, combinations of NheI, SpeI and NotI enzyme digests were used.
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Purified ARS1 plasmid DNA was covalently modified with biotin with Photoprobe biotin reagents (Vector Laboratories, USA) as manufacturer's instruction. Two hundred micrograms of biotinylated plasmid DNA was incubated with 6 mg streptavidin-coated paramagnetic beads (Dynal, USA) in a buffer containing 1 M NaCl, 10 mM HEPES-KOH (pH 7.6) and 1 mM EDTA at room temperature overnight. The beads were washed and equilibrated in a buffer containing 10 mM HEPES-KOH (pH 7.6) and 1 mM EDTA.
Preparation of yeast protein extract
Cells were grown in YEPD (2% polypeptone, 1% yeast extract, 2% glucose) or YEPGal (2% polypeptone, 1% yeast extract, 2% galactose) at 30 °C to about 2 x 107 cells/mL, arrested by the addition of -factor (Peptide Institute, Osaka, Japan) to 30 ng/mL and incubation for 3 h. Yeast whole cell extracts were prepared as previously described (Seki & Diffley 2000) with some minor modifications. Frozen cells were disrupted in a coffee mill with dry ice for 20 min as described or in the electronic mortar grinder (Retsch, Germany) with liquid nitrogen for 50 min. The protein concentration using the coffee mill is typically 50–80 mg/mL and the protein concentration using the mortar grinder is typically 70–110 mg/mL.
In vitro loading assay
The pre-RC assembly reaction (40 µL) contained 50 mM HEPES-KOH (pH 7.6), 625 mM sorbitol, 20 mM magnesium acetate, 0.125 mM EDTA, 5 mM EGTA, 2 mM DTT, 6 mM ATP, 40 mM creatine phosphate, 8 units creatine phosphokinase, 1.5 mg/mL poly(dI-dC)(dI-dC) (GE Healthcare, USA), 1% proteinase inhibitors (Sigma, USA), 1.1 pmol of either wild-type ARS1 or mutant ars1-DNA beads and 20 µL of protein extracts. The reaction mixture was incubated at 24 °C for 8 min. For multiple steps of loading reaction, ARS1-beads were washed twice in 0.4 mL of washing buffer (50 mM HEPES-KOH (pH 7.6), 10% glycerol, 150 mM potassium glutamate, 5 mM magnesium acetate, 1 mM EGTA, 1 mM DTT, 0.1% Triton X-100, and 2% proteinase inhibitors) and the second reaction mixture was prepared as above.
Antibodies
Rabbit anti-sera against Orc2p and Orc5p were as previously described (Kawasaki et al. 2000). Rabbit anti-sera against Abf1p and mouse antiserum against Cdc6p were gifts of John Diffley (Cancer Research UK, London Research Institute, London, UK). Rabbit antiserum against Cdt1p (expressed in and purified from E. coli) was prepared by a standard protocol. Antibodies against Mcm2p, Mcm5p, and Mcm7p were purchased from Santa Cruz Biochemistry (USA).
Purification of ORC, Cdt1p, and recombinant Cdc6p (rCdc6p)
ORC was purified from approximately 20 g of -factor-arrested ySC15 cells grown in YEPGal. After preparation of crude extracts, ORC was purified through sequential column chromatography basically as previously described (Donovan et al. 1999). Fractions that contain ORC were monitored by Western blotting using anti-Orc2 and anti-Orc5 serum. Approximately 80 µg ORC complex was obtained to near homogeneity. Cdt1p was purified from yeast YTS17 cells over-expressing Cdt1p. Twelve liters of logarithmically growing S. cerevisiae YTS17 cells in YEPGal were harvested and crude extracts were prepared as described (Seki & Diffley 2000). Cdt1p was purified through sequential column chromatography using 75 mL SP-Sepharose FF column, MonoQ (HR10/10), Mono S column (H 5/5), and Macro-Prep Ceramic Hydroxyapatite (Type I) (BIO-RAD) column (HR5/5). Fractions that contain Cdt1p were monitored by Western blotting using anti-Cdt1p serum. S. cerevisiae rCdc6p was purified from E. coli cells over-expressing His-tagged Cdc6p as previously described (Mimura et al. 2004).
Purification of Mcm2-7p
S. cerevisiae YHD124 cells were grown in 12 L YEPD to 2 x 107 cells/mL and arrested in G1 with 15 ng/mL -factor for 3 h, or arrested in G2/M by addition of 1.0 µg/mL Nocodazole for 3 h. For preparation of early S phase cells, -factor arrested YHD124 cells were harvested, washed with 2 L YEPR (2% Raffinose was used instead of Glucose) medium containing 100 µg/mL Pronase, resuspended into 12 L YEPR medium containing 50 µg/mL Pronase, and incubated at 23 °C for 2 h. Whole cell extracts were prepared as previously published (Seki & Diffley 2000) and Mcm2-7p was purified by TAP method as previously described (Puig et al. 2001). The eluted fractions from the calmodulin-beads were combined, concentrated and purified through a 5 mL 20–50% glycerol gradient (in 50 mM HEPES-KOH (pH 7.8), 100 mM potassium glutamate, 5 mM magnesium acetate, 0.02% NP-40 and 0.5 mM DTT) centrifugation at 45 000 r.p.m. and 4 °C for 24 h and proteins were analyzed by SDS-PAGE, followed by silver staining. Protein assignments were done by MALDI-TOF mass spectrometry (Hitachi Hitec, Japan). From the trace amount of proteins in the final purified product other than Mcm2-7p or Cdt1p, only truncated forms of Mcm2-7p or Cdt1p were identified.
DNA substrates for gel mobility shift assay
Six DNA oligonucleotides were synthesized and purified by HPLC (Table 2). The bubble substrate was assembled from two partially complementary oligonucleotides with "up" and "down" strand oligonucleotides (each 5 pmol) in a reaction (20 µL) containing 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 100 mM NaCl, and 1 mM DTT by heating at 95 °C for 5 min, followed by incubating at 65 °C for 5 min and slow cooling to 25 °C. Then, the "down" strand oligonucleotide was labeled at the 3' end with [-32P]dCTP and E. coli DNA polymerase I Klenow fragment in a reaction (30 µL) containing 50 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 100 mM NaCl, and 1 mM DTT by incubating at 25 °C for 30 min. The labeled DNA substrate was purified by polyacrylamide gel electrophoresis. Similarly, Y-fork, 5'-tail, 3' tail, and double stranded DNA substrates were prepared using 5'-(dT)50-4829 plus 3'-(dT)50-3829, 5'-(dT)50-4829 plus 5'-4829, 3'-4829 plus 3'-(dT)50-4829, and 3'-4829 plus 5'-4829, respectively. 3' end of 5'-(dT)50-4829 and 3'-4829 was labeled with radioactivity as above.
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Pre-RC assembly reaction from purified proteins was performed in basically same procedures as the "in vitro loading assay" described above with some modifications. BSA was added to 30 mg/mL, which greatly reduced the nonspecific binding of each protein to magnetic beads and non-ARS DNA. The reaction mixture was incubated at 24 °C for 10 min. When individual components were omitted, the reaction volume was made up with the appropriate buffer for each protein. To deplete ATP in the reaction mixture, 2.5 units of apyrase (Sigma, USA) was mixed.
Other methods
Western blotting followed by immunostaining with specific antibodies was carried out as previously described (Kawasaki et al. 2000). Quantification of the intensity of bands was performed with NIH image.
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Acknowledgements |
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Footnotes |
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Permanent address: aDepartment of Biotechnology and Bioinformatics, Chungbuk Provincial College of Science and Technology, 40 Geumgu-ri, Okcheon-eup, Okcheon-gun, Chungcheongbuk-do, 373-807, Republic of Korea
bThese authors contributed equally to this work.
* Correspondence: E-mail: ykawasak{at}fbs.osaka-u.ac.jp |
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Received: 2 February 2006
Accepted: 4 April 2006
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-S, and 20 fmol 32P-labeled bubble, Y-fork, 3'-tailed, 5'-tailed or double-stranded DNA substrates. After addition of 2 µL of 50% glycerol, the products were analyzed by 5% polyacrylamide gel electrophoresis, followed by autoradiography. Arrows indicate the positions of protein-DNA complex. (E) Seventy-five nanograms of Cdt1p-Mcm2-7p from G1, early S (S2), or G2/M-arrested cells was incubated at 30 °C for 30 min in the reaction (20 µL) containing 20 fmol 32P-labeled bubble- or Y-fork DNA substrates and 5 mM ATP or ATP-
