Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase cdk
1998; Springer Nature; Volume: 17; Issue: 19 Linguagem: Inglês
10.1093/emboj/17.19.5699
ISSN1460-2075
Autores Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle1 October 1998free access Xenopus Cdc45-dependent loading of DNA polymerase α onto chromatin under the control of S-phase cdk Satoru Mimura Satoru Mimura Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan Search for more papers by this author Haruhiko Takisawa Corresponding Author Haruhiko Takisawa Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan Search for more papers by this author Satoru Mimura Satoru Mimura Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan Search for more papers by this author Haruhiko Takisawa Corresponding Author Haruhiko Takisawa Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan Search for more papers by this author Author Information Satoru Mimura1 and Haruhiko Takisawa 1 1Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5699-5707https://doi.org/10.1093/emboj/17.19.5699 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info At the onset of S phase, chromosomal replication is initiated by the loading of DNA polymerase α onto replication origins. However, the molecular mechanisms for controlling the initiation are poorly understood. Using Xenopus egg extract, we report here the identification of a Xenopus homolog of Cdc45, a yeast protein essential for the initiation of replication, which is shown to be an essential molecule for the initiation of replication via the loading of DNA polymerase α onto chromatin. XCdc45, by physically interacting with the polymerase in the extract, became associated with chromatin only after nuclear formation. During S phase, XCdc45 co-localized with the polymerase in the nuclei, and the loading of the polymerase, which depended on endogenous XCdc45, was facilitated by exogenously added recombinant XCdc45. These findings, together with the apparent requirement of S-phase-cdk activity for the loading of XCdc45, suggest that XCdc45, under the control of S-phase cdk, plays a pivotal role in the loading of DNA polymerase α onto chromatin. Introduction In eukaryotes, the replication of the genome is strictly controlled during cell-cycle progression. One of the fundamental questions about the control of replication is how replication is initiated at the onset of S phase. This question is intimately related to another question: how is re-replication prohibited until the end of M phase? Recent studies have revealed that the latter mechanism, the licensing of replication, is conserved in eukaryotes (Kubota et al., 1997; Thömmes et al., 1997; Tanaka et al., 1997; also reviewed in Blow, 1996). The replication-competent, licensed chromatin is in a pre-replicative state (Diffley et al., 1994), which is formed by sequential assembly of origin recognition complex (ORC), Cdc6 and minichromosome maintenance protein complex (MCM) onto chromatin (Coleman et al., 1996; Romanowski et al., 1996; Rowles et al., 1996; Donovan et al., 1997; Tanaka et al., 1997). The assembly of MCM on the chromatin apparently plays an essential role for the licensing of replication, i.e. MCM is dissociated from chromatin as it is replicated, and MCM is re-associated with chromatin only at the end of M phase (reviewed in Chong et al., 1996; Kearsey et al., 1996). In contrast, how the licensed chromatin in the pre-replicative state is activated into a replicative state at the onset of S phase is poorly understood. The replicative state is established by recruiting DNA polymerase α-primase onto the replication origins, but little is known about what is the essential component for recruiting the polymerase and how these processes are controlled by S-phase-promoting kinases such as cyclin-dependent kinases (cdks). S-phase-promoting cdk has been proposed to play a dual role in controlling the initiation of chromosomal replication; one for triggering replication at the onset of S phase, and the other for preventing re-replication (Nasmyth, 1996; Stern and Nurse, 1996). Recent studies with budding yeast further suggest that S-phase-promoting cdk prevents re-replication by inhibiting the transition of replication origins to a pre-replicative state (Dahmann et al., 1995; Piatti et al., 1996). More recently, using formaldehyde cross-linking, it has been suggested that the cdk activity during S, G2 and M phases prevents MCM loading onto the replication origins (Tanaka et al., 1997). Similar data has also been reported using the cell-free extract of Xenopus eggs, showing that high levels of S-phase cdk2 prevent association of Mcm3, but not ORC, with chromatin (Hua et al., 1997). These data suggest that S-phase-cdk activity modulates the state of chromatin at the onset of S phase; however, the molecular nature of the target of these kinases has not been clarified. CDC45 of Saccharomyces cerevisiae was originally identified by screening cold-sensitive cdc mutants (Moir et al., 1982), which were subsequently shown to interact genetically with MCM5 (CDC46) and MCM7 (CDC47) (Hennessy et al., 1991). The cloning of CDC45 reveals that it is not a member of MCM, but is essentially required for the initiation of chromosomal replication (Hopwood and Dalton, 1996; Hardy, 1997; Zou et al., 1997). In addition, Cdc45 interacts genetically with ORC (Hardy, 1997; Zou et al., 1997), and physically with MCM proteins (Hopwood and Dalton, 1996; Dalton and Hopwood, 1997; Zou and Stillman, 1998) and the yeast replication origins (Aparicio et al., 1997). Therefore, Cdc45 was initially assumed to be required for the formation of the pre-replicative complex (Hopwood and Dalton, 1996) or as a component of the pre-replicative complex (Aparicio et al., 1997), which has been formed before START. However, a recent study indicates that Cdc45 functions in late G1, after START and prior to DNA synthesis (Owens et al., 1997). More recently, it has been shown that Cdc45 associates with chromatin after START, depending on Clb–Cdc28 kinase activity, thus suggesting that Cdc45 associates with the pre-replicative complex after activation of S-phase-promoting cdk (Zou and Stillman, 1998). All of these characteristics make Cdc45 a good candidate for involvement in the activation of the licensed chromatin, in the pre-replicative state. However, its exact role in initiation of replication remains largely unknown. Here, we report the identification of a Xenopus Cdc45 homolog as an essential molecule for the loading of DNA polymerase α onto chromatin under the control of S-phase-cdk activity. Results Identification of a Xenopus Cdc45 homolog A potential Xenopus Cdc45 homolog (XCdc45) was cloned as follows. We first identified a partial sequence of a putative human homolog (EST64671) by searching for the homolog of yeast Cdc45 in the EST database. Next we cloned a full-length human cDNA (DDBJ/EMBL/GenBank accession No. AF062495) by screening a HeLa cDNA library with the EST sequence as a probe. Finally, we isolated a putative Xenopus homolog (DDBJ/EMBL/GenBank accession No. AF062494) by screening a Xenopus oocyte cDNA library with the full-length human cDNA as a probe. The open reading frame of the Xenopus cDNA encoded a protein consisting of 567 amino acids with a calculated molecular mass of 65 kDa. The predicted amino acid sequence showed a strong homology to the putative human homolog (75% identity) and significant sequence identity with the yeast Cdc45 (∼25%). Highly conserved regions among these three protein sequences (Figure 1A) were noted at locations along the entire length of the peptides. In addition, acidic patches (Hopwood and Dalton, 1996), consisting of ∼50% of the acidic amino acids, and potential nuclear localization sequences located at the end of the patches (Hopwood and Dalton, 1996), were also found to be conserved among these proteins. These data suggest that the cloned Xenopus and human sequences are indeed putative homologs of the yeast Cdc45. Figure 1.Requirement of the Xenopus Cdc45 homolog for the replication of chromosomal DNA in cell-free extracts of Xenopus eggs. (A) The schematic structure of a putative Xenopus Cdc45 homolog compared with S.cerevisiae Cdc45 and a putative human homolog. Numerical values indicate the percent identity of amino acids in each conserved region between XCdc45 and the other proteins. The percentages of acidic amino acids are indicated just below the acidic patch region. (B) Expression of endogenous and recombinant XCdc45 protein. Endogenous protein was isolated by immunoprecipitating S-phase egg extract with anti-XCdc45 antibody bound to a protein A matrix. The first immunoprecipitates contained most of XCdc45 proteins. Recombinant XCdc45 protein tagged with His6 at the C-terminus (XCdc45-His6) was expressed in Sf9 cells and purified with Ni–NTA resin. Both samples were resolved by SDS–PAGE, and visualized by Coomassie Blue staining. (C) Immunodepletion of XCdc45 from S-phase egg extract. Extracts were treated with preimmune (mock) and anti-XCdc45 antibodies bound to protein A matrix, resolved by SDS–PAGE and immunoblotted with anti-XCdc45 C-terminal peptide antibody. (D) Inhibition of the replication activity of S-phase egg extract upon depletion of XCdc45. Sperm chromatin was incubated in mock, mock-plus 10 μg/ml aphidicolin and XCdc45-depleted extracts for 90 min at 23°C. Replication activity was monitored as the incorporation of 32P-labelled dCTP into DNA. Relative values are given. (E) Requirement of XCdc45 for the initiation stage of DNA replication. Sperm chromatin was incubated in the extracts as in (D), and the total DNA was isolated and subjected to alkali agarose gel electrophoresis. Download figure Download PowerPoint Involvement of XCdc45 in DNA replication To investigate the role of the putative XCdc45 in DNA replication, we expressed a His6-tagged version of XCdc45 (XCdc45-His6) in baculovirus-infected Sf-9 cells. The recombinant protein, purified with Ni-nitrilotriacetic acid (NTA) agarose, consisted of a single polypeptide with an apparent molecular mass of 66 kDa (Figure 1B). The purified XCdc45-His6 protein and glutathione-S-transferase (GST)-fused C-terminal peptide were used as antigens to produce polyclonal antibodies. Both antibodies recognized a 63 kDa protein in S-phase egg extract (Figures 1C and 3A). For immunoprecipitation of endogenous XCdc45 from the extract, the antisera were further affinity purified with the recombinant protein. Figure 1B shows that the first immunoprecipitates of the extract with the purified anti-XCdc45 antibody yielded a specific protein with an apparent molecular mass of 63 kDa, slightly smaller than that of the recombinant protein which contained an additional tag. The immunoprecipitated 63 kDa protein was also recognized by antibodies against the full-length (Figure 3A) and C-terminal peptide (data not shown). These results indicate that the protein corresponding to the cloned Xenopus sequence is present in the egg extract. Figure 2.Identical behavior of XCdc45 and DNA polymerase α throughout the S phase. (A) Co-immunoprecipitation of DNA polymerase and XCdc45 in S-phase egg extract with anti-XCdc45 antibody. The egg extract was incubated with preimmune, anti-XCdc45 and anti-XMcm3 antibodies bound to a protein A matrix. The egg extract (1 μl) and immunoprecipitates from 50 μl extracts were resolved by SDS–PAGE and immunoblotted with anti-DNA polymerase α, anti-XMcm5 and anti-XCdc45 antibodies. (B) Simultaneous loading of XCdc45 and polymerase onto chromatin during nuclear formation. Sperm chromatin was incubated in the egg extract at 23°C for the indicated times and the chromatin fraction was isolated, resolved with SDS–PAGE, and immunoblotted with anti-DNA polymerase α, anti-XMcm6 and anti-XCdc45 antibodies. (C) Co-localization of XCdc45 and the polymerase in replicating nuclei. Sperm chromatin was incubated in the egg extract for 30 and 60 min at 23°C in the presence and absence of 10 μg/ml aphidicolin, and the samples were treated with Triton X-100 followed by fixation with formaldehyde, and processed for immunofluorescence. Scale bar, 20 μm. (D) Different behavior of XCdc45 and XMcm6 in replicating nuclei. Sperm chromatin was incubated in the egg extract for 30, 40 and 90 min, and the samples were treated and processed as for (C). Scale bar, 20 μm. Download figure Download PowerPoint With the anti-XCdc45 antibody, endogenous XCdc45 could be depleted from the extract almost completely (Figure 1C). We first examined whether or not XCdc45 is required for DNA replication in the extract. Upon depletion of XCdc45 from the extract, the replication activity was diminished to <10% of the mock-depleted extract (Figure 1D). In order to examine the nature of residual replication activity, total DNA was isolated from the extracts and subjected to alkali agarose gel electrophoresis (Figure 1E). The detection of replicated DNA with the incorporation of 32P-labelled dCTP showed that nascent DNA was synthesized in the mock-depleted extract in the presence of a low concentration of aphidicolin (∼80% inhibition of total DNA synthesis). In the XCdc45-depleted extract, no nascent DNA was synthesized, but a small amount of DNA with high molecular mass was produced. The high molecular DNA was also produced in the mock-depleted extract as a result of a complete round of replication (compare with Mahbubani et al., 1997). In accord with this, the polymerase activity measured with activated DNA was not significantly decreased upon XCdc45 depletion (data not shown). These results suggest that XCdc45 is required for the initiation stage of DNA replication in the extract. In order to obtain more insight into the role of XCdc45, we examined the behavior of XCdc45 in the egg extract. Upon addition of sperm chromatin to the extract, ORC and MCM became associated with the chromatin within 10 min, before the nuclear structures had been assembled around the chromatin. However, XCdc45 only became associated with chromatin 30 min after the addition of chromatin; after the formation of nuclei. In the absence of membrane fractions of the extract (high speed supernatant; HSS), we detected neither the formation of nuclei nor the association of XCdc45 with the chromatin (Figure 2A), suggesting that nuclear formation is required for the assembly of XCdc45 onto chromatin. Essentially the same results were obtained by examining the chromatin fractions by immunoblotting (Figure 2B). XMcm6 became associated with chromatin before, or in the absence of, nuclear formation. As with the behavior of XCdc45, we found that XCdc45 is not required for the formation of licensed chromatin (Figure 2B). Examination of chromatin fractions prepared from mock- and XCdc45-depleted extracts demonstrated no difference in the association of XOrc1 and XMcm3 with the chromatin, while XCdc45 was only absent in the chromatin fraction prepared from the depleted extract. These results suggest that XCdc45 is not required for licensing chromatin, and that it is assembled onto the licensed chromatin only after nuclear formation. Consistent with this, recombinant XCdc45 could rescue the replication activity of the depleted extract when added after nuclear formation as well as before its formation (Figure 2C). These results indicate that recombinant XCdc45 could be functionally substituted for endogenous XCdc45 and that XCdc45 is required for the initiation process after the formation of nuclear structures. Figure 3.Loading of XCdc45 onto chromatin after nuclear formation. (A) Immunofluorescent detection of XCdc45 and XMcm6 on chromatin with and without nuclear formation. Sperm chromatin was incubated in S-phase egg extract (low speed supernatant; LSS), and membrane-depleted S-phase extract (high speed supernatant; HSS) at 23°C for the indicated times, and the localization of XCdc45 and XMcm6 was visualized by rabbit anti-XCdc45 and mouse anti-XMcm6 antibodies followed by Cy2-labelled anti-rabbit and Texas red-labelled anti-mouse antibodies, respectively. DNA was visualized with Hoechst. Scale bar, 20 μm. (B) Loading of XCdc45 onto the chromatin after the assembly of licensed chromatin. Sperm chromatin was incubated in egg extracts as for (A). As a control (−sperm), sperm chromatin was omitted from the extract, and mock-, and XCdc45-depleted extracts were prepared as for Figure 1C. The chromatin fractions isolated from the extracts were resolved by SDS–PAGE, and visualized by immunoblotting with specific antibodies as indicated. (C) Rescue of the replication activity of XCdc45-depleted extract with recombinant XCdc45. Sperm chromatin was incubated in mock- and XCdc45-depleted extracts in the presence of [32P]dCTP, and XCdc45-His6 (20 ng/μl extract) was added to the XCdc45-depleted extracts before (0 min) or after nuclear formation (45 min). Sixty and 90 min after the addition of sperm chromatin, the replication reaction was stopped and DNA was isolated and subjected to agarose gel electrophoresis. Download figure Download PowerPoint Investigation into the interaction of XCdc45 with polymerase Nuclear formation, which is essential for replication (Blow and Laskey, 1986; Newport, 1987), leads to the accumulation of various proteins required for replication. In order to understand the function of XCdc45, we looked for possible target proteins which physically interact with XCdc45 by examining the immunoprecipitates from the extract. Figure 3A shows that a small amount of DNA polymerase α was co-immunoprecipitated with XCdc45, but not the control and anti-XMcm3 immunoprecipitates. We also examined the possible association of XMcm7 and PCNA with XCdc45, but failed to detect a significant association (data not shown). It should also be noted that a very faint signal of XMcm5 was detected with anti-XCdc45 immunoprecipitates. This contrasts with the previous reports from budding yeast, which showed that the yeast Cdc45 forms a physical complex with Mcm5 and 7 in the cell extracts prepared from log-phase cultures (Hopwood and Dalton, 1996; Dalton and Hopwood, 1997). These conflicting results may be explained if XCdc45 can form a complex with XMcms only after nuclear formation, i.e. at the onset of S phase. A recent report with budding yeast has shown that Cdc45 physically interacts with Mcm2 during S phase (Zou and Stillman, 1998). The possible association of XCdc45 with polymerase led us to speculate that XCdc45 is required for replication by physically interacting with the polymerase to recruit it onto replication origins. If this were the case, XCdc45 should be loaded onto the chromatin at the same time or prior to the loading of the polymerase. Immunoblotting of the chromatin fractions showed that the polymerase and XCdc45 became associated with chromatin at the same time, i.e. after the formation of the nuclei (Figure 3B). Figure 3C shows further that XCdc45 and the polymerase were co-localized in the nuclei throughout the progression of S phase. Both proteins precisely co-localized in every nuclei formed in the extract at the onset of replication (30 min). During the progression of S phase, both signals in the nuclei decreased (60 min), and essentially no signal was detected 90 min after nuclear formation, when the replication was completed in most of the nuclei (compare with Figure 3D). Conspicuous co-localization of XCdc45 and the polymerase was also observed when the replication was inhibited by aphidicolin. Both signals in the nuclei were enhanced in the presence of aphidicolin (Figure 3C). This may suggest that the activation of origins in the absence of DNA synthesis facilitates the accumulation of both proteins onto the chromatin. In contrast to the polymerase, the XMcm6 signal in the nuclei was most intense at the onset of replication, and the signal rapidly decreased during replication (Figure 3D). The intensity of the XCdc45 signal in the nuclei increased after the start of replication (40 min), and its intensity was reciprocally related to that of XMcm6. These results showed that XCdc45 function is closely related to the loading of the polymerase onto chromatin. An essential requirement of XCdc45 for the loading of the polymerase is shown in Figure 4A. Upon depletion of XCdc45 from the extract, neither the loading of the polymerase nor the initiation of replication was observed (see Figure 1D). In contrast, the loading of Mcm proteins onto the chromatin occurred normally in the depleted extracts (Figure 2B). These results suggest that XCdc45 is required for the loading of the polymerase on the licensed, pre-replicative chromatin, which has been assembled before nuclear formation. Data with recombinant XCdc45 provided further support for the idea that XCdc45 is intimately involved in the loading of the polymerase. Figure 4B shows that the addition of an excess of recombinant XCdc45 (the concentration of endogenous XCdc45 was estimated to be about 10 ng/μl extract), accelerated the association of the polymerase with chromatin in parallel with the rapid association of the recombinant XCdc45 on chromatin. In addition, the rapid association of the recombinant XCdc45 apparently suppressed the association of endogenous XCdc45 on the chromatin (Figure 4C), suggesting competition for the same site. Therefore, the acceleration of the polymerase loading with the recombinant protein suggests that XCdc45 is essential for the rate determining step in the polymerase loading. Figure 4.Essential role of XCdc45 for the loading of DNA polymerase α onto the chromatin. (A) Requirement of XCdc45 for the loading of polymerase onto chromatin. Sperm chromatin was incubated in the mock- and XCdc45-depleted extracts for 40 min at 23°C, and the samples were processed for immunofluorescence as for Figure 3C. Scale bar, 20 μm. (B and C) Stimulation of the loading of the polymerase onto chromatin by recombinant XCdc45. Sperm chromatin was incubated in S-phase egg extract with 0, 25 and 100 ng recombinant XCdc45-His6 per μl extract for 30 min at 23°C, and samples were processed for immunofluorescence (B), and immunoblotting of the chromatin fraction (C). R and E denote recombinant and endogenous XCdc45, respectively. Scale bar, 20 μm. Download figure Download PowerPoint S-phase cdk-dependent loading of XCdc45 onto chromatin The next question is how the loading of XCdc45 onto the chromatin might be regulated. Since nuclear formation has been shown to facilitate the accumulation of S-phase cdk into the nuclei (Hua et al., 1997), we examined whether the cdk activity is required for the loading of XCdc45. With Xenopus egg extracts, it has been established that p21(Cip1), inhibits replication through the inhibition of S-phase-cdk activity in the extract (Strausfeld et al., 1994; Chen et al., 1995; Jackson et al., 1995). The same concentration of p21 sufficient to inhibit replication also inhibited the loading of both XCdc45 and the polymerase onto the chromatin (Figure 5A). Immunoblotting of chromatin fractions confirmed further that the loading of XOrc1 and XMcm3 was not affected by p21 (Figure 5A), consistent with the fact that both proteins were loaded before the formation of nuclei. This observation is in agreement with the previous finding that the pre-replicative complex is formed in the absence of the S-phase-cdk activity (Dahmann et al., 1995; Hua et al., 1997; Tanaka et al., 1997). In order to confirm that the effect of p21 is due to the inhibition of cdk activity, we next prepared an extract depleted of Xcdk2, an essential component for the initiation of replication in the extract (Fang and Newport, 1991; Jackson et al., 1995). In order to do this, we used suc1-conjugated agarose, which was successfully used to deplete Xcdc2 and Xcdk2 from the extract (Blow and Nurse, 1990; Fang and Newport, 1991; Strausfeld et al., 1996). Upon treating the extract with suc1 agarose, Xcdk2 was almost completely depleted from the extract, while a small amount of Xcdc2 remained in the extract (Figure 5B), and both the loading of XCdc45 and the polymerase onto the chromatin were severely inhibited (Figure 5C). All these results indicate that S-phase cdk is required for the loading of XCdc45, and eventually for the loading of the polymerase onto the pre-replicative chromatin. Figure 5.Requirement of S-phase-cdk activity for loading of XCdc45 onto chromatin. (A) Inhibition of the loading of XCdc45 and DNA polymerase α onto chromatin by the cdk inhibitor, p21. Sperm chromatin was incubated in S-phase egg extract in the presence and absence of 300 nM p21 for 30 min (immunofluorescence, IF) and 45 min (immunoblotting, IB) at 23°C, and the samples were processed for immunofluorescence with anti-XCdc45 and anti-DNA polymerase α antibodies (IF), and for immunoblotting of the chromatin fraction with anti-polymerase, -XOrc1, -XMcm3 and -XCdc45 antibodies (IB). Scale bar, 20 μm. (B) Depletion of cdks from S-phase egg extract with suc1 agarose. The egg extracts were treated with mock- and suc1-conjugated agarose, and the treated extract together with the proteins bound to the agarose beads were resolved by SDS–PAGE and immunoblotted with anti-PSTAIR antibody. (C) Absence of the loading of XCdc45 and the polymerase onto chromatin following depletion of cdks. Sperm chromatin was incubated in the egg extracts treated with mock- and sucl agarose for 30 min at 23°C, and XCdc45 and the polymerase were detected by immunofluorescence. Scale bar, 20 μm. Download figure Download PowerPoint Discussion In the present study we have identified Xenopus Cdc45 homolog as an essential molecule for the initiation of chromosomal replication in the extract of Xenopus eggs. XCdc45 became associated with chromatin only after nuclear formation, and it could rescue the replication activity of the XCdc45-depleted extract after nuclear formation. This contrasts with the behavior of XMCM, XCdc6 and XORC, which assemble with chromatin before nuclear formation (Coleman et al., 1996; Romanowski et al., 1996; Rowles et al., 1996). In addition, XMCM and XCdc6 are unable to rescue the replication activity of the depleted extracts after nuclear formation (Coleman et al., 1996; Kubota et al., 1997). These data suggest that XCdc45 is not involved in the licensing reaction observed with the extract, and in accordance with this, XCdc45 is not required for the loading of XMCM onto chromatin. Therefore, it is reasonable to assume that XCdc45 is not a component of the licensed chromatin in a pre-replicative state. Absence of physical interaction between XCdc45 and XMCM in the egg extract further suggests that XCdc45 interacts with XMCM only after nuclear formation. Actually, the inhibition of replication activity with aphidicolin led to the co-localization of XCdc45 and XMCM in the nuclei, since both proteins appeared to be uniformly distributed on the chromatin in the absence of replication activity. Preliminary experiments indicated the physical association of XMCM and XCdc45 under such conditions (data not shown). In consideration of recent data on Cdc45 (Zou and Stillman, 1998), we would like to propose that XCdc45 interacts with XMCM on the licensed chromatin after nuclear formation, that is, at the onset of S phase. The present finding that XCdc45 is required for the loading of DNA polymerase α reinforced the view that XCdc45 plays a pivotal role in the activation of the licensed chromatin. Previous studies of the simian virus 40 (SV40) DNA replication system (Challberg and Kelly, 1989; Stillman, 1989; Borowiec et al., 1990; Waga and Stillman, 1994) provide us with a good framework for understanding the molecular mechanisms for the initiation of replication in eukaryotes. In the SV40 system, SV40 large T antigen plays crucial roles in the initiation (Fanning and Knippers, 1992). It has three distinct functions: first to recognize origins, secondly to unwind the origin DNA, and thirdly to recruit the polymerase onto the origins. Recent studies with eukaryotic chromosome replication reveals that the first function is provided by ORC, a conserved initiator protein for the assembly of pre-replicative complex (Stillman, 1996; Dutta and Bell, 1997). The second function may be provided by the MCM 4-6-7 complex, which has recently been reported as having DNA helicase activity (Ishimi, 1997). The present study suggests that the third function requires a Cdc45 homolog as an essential component. This notion is further supported by recent studies concerning the physical association of Cdc45 with replicating origins of the yeast S.cerevisiae (Aparicio et al., 1997). Although the dependence of the polymerase loading on XCdc45 did not support the direct role of XCdc45 on the polymerase loading, the physical association of XCdc45 and the polymerase, together with the close relationship between the loading of XCdc45 and the polymerase, suggests that XCdc45 mediates the loading of the polymerase, possibly as a component of a putative loading protein of the polymerase. The present study further shows that the loading of XCdc45 onto chromatin is under control of S-phase cdk. An essential role for cdk in the regulation of cell-cycle progression has been established (Nasmyth, 1996; Stern and Nurse, 1996). However, the target of the kinase at the onset of S phase has not been identified. Here, we have shown that the loading of XCdc45 is apparently controlled by S-phase-cdk activity, possibly by cdk2 in the extract. It is therefore possible that factor
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