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• W2165749026 abstract "Article20 March 2012free access Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components Mai Kanke Mai Kanke Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Yukako Kodama Yukako Kodama Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Tatsuro S Takahashi Tatsuro S Takahashi Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Takuro Nakagawa Takuro Nakagawa Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Hisao Masukata Corresponding Author Hisao Masukata Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Mai Kanke Mai Kanke Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Yukako Kodama Yukako Kodama Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Tatsuro S Takahashi Tatsuro S Takahashi Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Takuro Nakagawa Takuro Nakagawa Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Hisao Masukata Corresponding Author Hisao Masukata Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Author Information Mai Kanke1, Yukako Kodama1, Tatsuro S Takahashi1, Takuro Nakagawa1 and Hisao Masukata 1 1Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan *Corresponding author. Department of Biological Sciences, Graduate School of Science, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan. Tel.: +81 6 6850 5432; Fax: +81 6 6850 5440; E-mail: [email protected] The EMBO Journal (2012)31:2182-2194https://doi.org/10.1038/emboj.2012.68 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function. Introduction Initiation of DNA replication requires replicative DNA helicase, which unwinds the origin DNA duplex to provide single-stranded DNA templates for replicative DNA polymerases. In eukaryotes, the heterohexameric mini-chromosome maintenance 2-7 (Mcm2-7) complex comprises the core of replicative DNA helicase (Bell and Dutta, 2002). Current evidence indicates that two additional factors, Cdc45 and the GINS (go–ichi–ni–san) complex, are essential stoichiometric components of replicative DNA helicase (Takahashi et al, 2005; Moyer et al, 2006; Pacek et al, 2006; Bochman and Schwacha, 2009; Ilves et al, 2010). It has been shown that Cdc45, Mcm2-7 and GINS form a stable ‘CMG’ complex (Cdc45–Mcm2-7–GINS) that translocates on a single-stranded DNA in the 3′–5′ direction (Moyer et al, 2006; Ilves et al, 2010). Chromatin loading and activation of the replicative DNA helicase are tightly controlled by progression of the cell cycle. In G1-phase, when CDK activity is low, the Mcm2-7 complex is loaded onto replication origins by the origin recognition complex, Cdc6/Cdc18 and Cdt1, forming the pre-replicative complex (pre-RC) (Bell and Dutta, 2002). At this stage, a head-to-head double-hexameric Mcm2-7 encircles the origin DNA duplex as a helicase-inactive form (Evrin et al, 2009; Remus et al, 2009). At the onset of S-phase, when the CDK activity arises, a number of replication factors act on the pre-RCs, converting the helicase-inactive pre-RCs into the CMG helicases. The Dbf4-dependent Cdc7 kinase (DDK) phosphorylates the Mcm2-7 complex and stimulates loading of Sld3 onto pre-RCs (Yabuuchi et al, 2006; Heller et al, 2011). CDK phosphorylates Sld3 and Sld2/Drc1, promoting formation of a complex of Sld3–Dpb11/Cut5–Sld2 at replication origins (Masumoto et al, 2002; Tanaka et al, 2007; Zegerman and Diffley, 2007; Fukuura et al, 2011). Finally, these factors collectively recruit GINS and Cdc45 onto the Mcm2-7 complex, resulting in full assembly of the CMG helicase (Remus and Diffley, 2009; Araki, 2010). Requirement of Sld3, Sld2 and Dpb11 for initiation of DNA replication is likely limited at the assembly step of the CMG helicase (Kanemaki and Labib, 2006; Labib, 2010; Taylor et al, 2011). Origin DNA is unwound by the activated replicative helicase, and then RPA, a eukaryotic single-stranded DNA-binding protein complex, and DNA polymerases are recruited to origins, resulting in assembly of complete replisomes. Recent experiments have strongly suggested that a single replisome contains a single CMG complex that translocates on the leading strand template in the 3′–5′ direction (Gambus et al, 2006; Moyer et al, 2006; Ilves et al, 2010; Yardimci et al, 2010; Costa et al, 2011; Fu et al, 2011). These findings argue that activation of the replicative DNA helicase involves dynamic structural changes of the Mcm2-7 complex from the double-stranded DNA (dsDNA)-bound double hexamer to the single-stranded DNA (ssDNA)-bound single hexamer. Mcm10/Cdc23 is an evolutionally conserved protein essential for DNA replication (Nasmyth and Nurse, 1981; Aves et al, 1998). It was identified through genetic screenings for DNA replication defects (Dumas et al, 1982; Solomon et al, 1992) and mini-chromosome maintenance defects (Maine et al, 1984; Merchant et al, 1997). The central domain of Mcm10, which contains a zinc-finger motif, is required for cell viability in budding yeast (Cook et al, 2003), and is highly conserved among eukaryotes (Homesley et al, 2000; Izumi et al, 2000). In vitro studies have shown that purified Mcm10 binds to both dsDNA and ssDNA in human, Xenopus, budding yeast and fission yeast (Fien et al, 2004; Okorokov et al, 2007; Robertson et al, 2008; Warren et al, 2008, 2009; Eisenberg et al, 2009). Although Mcm10 has no sequence homology to Mcm2-7, it physically interacts with the members of the Mcm2-7 complex (Merchant et al, 1997; Homesley et al, 2000; Hart et al, 2002; Lee et al, 2003). Over-expression of Mcm10 partially suppresses the cold sensitivity of the nda4-108/mcm5 mutant (Hart et al, 2002). Double mutants of mcm10 with mcm2-7 mutants exhibit enhanced temperature sensitivity, synthetic lethality or suppression of temperature sensitivity, depending on the mutation alleles (Homesley et al, 2000; Liang and Forsburg, 2001; Hart et al, 2002; Lee et al, 2010). These physical and genetic interactions imply that Mcm10 functions in close coordination with Mcm2-7. The exact step, at which Mcm10 functions, in the initiation of DNA replication remains enigmatic. It has been reported that Mcm10 is required for recruitment of Cdc45 to activate the Mcm2-7 helicase in budding yeast, fission yeast and Xenopus egg extracts (Wohlschlegel et al, 2002; Gregan et al, 2003; Sawyer et al, 2004). On the other hand, other studies have shown that depletion of Mcm10 does not affect the chromatin association of Cdc45 either in vivo or in vitro (Ricke and Bielinsky, 2004; Heller et al, 2011). Mcm10 has also been shown to be required for tethering DNA polymerase α (Pol α) at replication forks, and for controlling the stability of the catalytic subunit of Pol α in budding yeast and human cells (Ricke and Bielinsky, 2004, 2006; Chattopadhyay and Bielinsky, 2007), whereas siRNA for Mcm10 in human cells does not affect the stability of Pol α (Zhu et al, 2007). Therefore, although these observations consistently suggest that the function of Mcm10 is closely related to the CMG complex and the components of the replisome, the molecular function of Mcm10 is not clearly understood. To uncover the role of Mcm10 in initiation of DNA replication, we applied a conventional promoter shut-off system combined with a recently developed auxin-inducible protein degradation system (off-aid) to Mcm10 (Nishimura et al, 2009; Kanke et al, 2011). The off-aid depletion efficiently removed >99% of Mcm10 from cells, allowing us to determine precisely the step at which Mcm10 executes its essential function. Interestingly, Mcm10 was required for origin DNA unwinding after assembly of the CMG components on replication origins. In addition, initiation-specific factors, such as Sld3, Cut5 and Drc1, were not released from origins in the absence of Mcm10. The zinc finger of Mcm10, whose mutations decreased the self-interaction and ssDNA-binding activity of Mcm10, was essential for origin unwinding but not for association of Mcm10 onto replication origins. Our results demonstrate that Mcm10 plays a novel key role, together with the CMG components, in the origin DNA unwinding step via its zinc-finger motif for initiation of DNA replication. Results Mcm10 is not required for loading of the CMG components onto replication origins Assembly of the CMG components at replication origins is a key step for initiation of DNA replication. We first examined whether Mcm10 is required for this process. Because the existing mutant allele of Mcm10 was leaky and not suitable for analysis at the molecular level (Liang and Forsburg, 2001), we adopted an approach involving depletion of Mcm10 protein from living cells. For efficient and tight depletion of the protein, we combined an auxin-inducible degron (AID) system (Nishimura et al, 2009; Kanke et al, 2011) with the thiamine-repressible nmt81 promoter (Pnmt81) (Pnmt81-mcm10-aid: mcm10-off-aid). mcm10-off-aid cells were grown in the presence of thiamine for 14 h to repress mcm10 transcription, and then synchronized using the temperature-sensitive mutation of cdc25-22, which causes cell-cycle arrest at the G2/M boundary. Auxin was added 1 h prior to G2/M release into 25°C (see Figure 1 legends). Immunoblotting of cell extracts showed that the Mcm10-aid protein was decreased to as little as 0.5% of the amount in wild-type (WT) cells, corresponding to <18 molecules per cell at the time of G2/M release (Figure 1A; Supplementary Figure S1). We analysed the DNA contents of the cells by flow cytometry to examine the defect in DNA replication. Cells without depletion generated a 4C DNA peak, indicative of DNA replication, because cytokinesis occurs during the period corresponding to S-phase in the normal fission yeast cell cycle (Figure 1B, left). When non-depleted cells were treated with hydroxyurea (HU), which depletes the dNTP pool, DNA replication was blocked at an early stage, generating cells with 1C DNA content (Figure 1B, middle). Mcm10-depleted cells generated a sharp 1C peak similar to HU-arrested cells (Figure 1B, right), indicating arrest of the cell cycle in the early stage of DNA replication. Figure 1.Localization of the CMG components at replication origins in Mcm10-depleted cells. (A) The mcm10-off-aid cells carrying psf2–flag and cdc45–myc were incubated with thiamine for 14 h and arrested at the G2/M boundary by incubation at 36°C for 3.5 h. Auxin (0.5 mM) was added 1 h before release from G2/M block to 25°C. HU (12 mM) was added to the cells without depletion. The amounts of proteins from asynchronous cells (asy, lane 1), before G2/M arrest (thia 14 h, lane 2) and at the indicated time points after release from the G2/M boundary (lanes 3–6) were analysed by immunoblotting using anti-Mcm10 and anti-α-tubulin antibodies. An arrowhead and asterisks (*) indicate the positions of Mcm10-aid and non-specific bands, respectively. (B) DNA contents of non-depleted cells with (middle) or without (left) HU treatment and Mcm10-depleted cells (right) were analysed by flow cytometry. (C–E) DNA fragments immunoprecipitated with Mcm6 (C), Psf2–Flag (D) and Cdc45–myc (E) were analysed by real-time PCR using primer sets for two early origins, ars2004 (blue) and ars3002 (cyan), and for the non-origin region, nonARS1 (grey). The columns indicate IP recovery (%) ±s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S2. Download figure Download PowerPoint Under Mcm10-depleted conditions, we examined the origin localization of Mcm6, Psf2 (GINS) and Cdc45 by ChIP assay. DNA immunoprecipitated with these factors was quantified by real-time PCR for the ars2004 and ars3002 loci, which are efficient replication origins on chromosomes II and III, respectively, and nonARS1, located 30 kb distant from ars2004. In HU-arrested cells without depletion, IP recovery of the two origins with Mcm6, Psf2 and Cdc45 was similar to that of nonARS1 at G2/M release (0 min), but was increased at 85 and 100 min (Figure 1C–E, left), showing that these factors were localized at the origins in S-phase. In Mcm10-depleted cells, Psf2 and Cdc45, as well as Mcm6, bound to the origins, as was the case in HU-treated cells (Figure 1C–E, right). These results show that Mcm10 is not required for origin localization of the CMG components, and suggest that Mcm10 functions downstream of recruitment of the CMG components. Mcm10 recruitment to replication origins is dependent on the CMG components Next, we examined the dependency of Mcm10 recruitment. If origin loading of Mcm10 was dependent on the CMG components, then depletion of GINS or Cdc45 would impair Mcm10 recruitment. We depleted Psf1 or Cdc45 using the off-aid system as described for Mcm10 depletion and in the previous study (Kanke et al, 2011). The psf1-off-aid or cdc45-off-aid cells were incubated with thiamine for transcriptional repression, and auxin was added 1 h prior to G2/M release at 25°C. The amount of Psf1 or Cdc45 after depletion was decreased to <1% of the original amount, and the cells were arrested with unreplicated DNA (Supplementary Figure S3A–F). Under these conditions, localization of Mcm6 and Mcm10 was examined by ChIP assay. In HU-treated cells without depletion, Mcm6 was preferentially localized at ars2004 and ars3002 in S-phase (Figure 2A and C, left). Mcm10, which was not localized at the origins at the G2/M boundary (0 min), was detected at the origins in S-phase (Figure 2B and D, left). In Psf1-depleted or Cdc45-depleted cells, however, Mcm10 was not significantly detected at the origins, whereas Mcm6 accumulated (Figure 2A–D, right). To examine whether Mcm10 localization is dependent on a factor that is recruited to origins after assembly of the CMG complex, we depleted Pol α, which binds to chromatin depending on RPA (Walter and Newport, 2000). The catalytic and primase subunits of Pol α (Pol1 and Spp2, respectively) were depleted using the off-aid system, resulting in cell-cycle arrest at the early stage of DNA replication (Supplementary Figure S3G and H). The results of ChIP assay showed that Mcm10 bound to the origins in Pol α-depleted cells (Figure 2F, right). These results indicate that GINS and Cdc45, but not Pol α, are required for recruitment of Mcm10 to replication origins, suggesting that Mcm10 is recruited after loading of the CMG components on replication origins. Figure 2.Mcm10 binds to replication origins depending on GINS and Cdc45, but not on Pol α. psf1-off-aid cells and cdc45-off-aid cells carrying flag–mcm10 were incubated with thiamine (10 μg/ml) for 4 h and then arrested at the G2/M boundary by incubation at 36°C for 3.5 h. pol1-off-aid spp2-off-aid cells carrying flag–mcm10 were incubated with thiamine (10 μg/ml) for 6 h and then arrested at the G2/M boundary. Auxin (0.5 mM) was added 1 h before release. The cells were released from G2/M block and HU (12 mM) was added to them without depletion. DNA immunoprecipitated with Mcm6 and Flag–Mcm10 in psf1-off-aid cells (A, B), cdc45-off-aid cells (C, D), or pol1-off-aid spp2-off-aid cells (E, F) at the indicated time points was assayed with a real-time PCR system. Columns indicate IP recovery (%) ±s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S4. Download figure Download PowerPoint The CMG components form a complex in the absence of Mcm10 It has been reported that the CMG components form a complex that is stable in the presence of a high salt concentration (Gambus et al, 2006). We examined whether the CMG components recruited at the origin in the presence or absence of Mcm10 formed a complex. By immunoprecipitation of Psf2–Flag from HU-treated cell extracts, in which genomic DNA was digested by DNase I, Mcm6 and Cdc45–myc were co-precipitated in the presence of 200 mM NaAc, while these proteins were hardly detected in the precipitates from non-tagged cells (Figure 3A, lanes 3 and 4). Co-precipitation of Mcm6 and Cdc45–myc with Psf2–Flag was specific in S-phase (100 min) but not in G2/M-phase (0 min) cells (Figure 3B, lanes 5 and 6). In Mcm10-depleted cells, Mcm6 and Cdc45–myc were co-precipitated with Psf2–Flag, in amounts similar to those in HU-treated cells without depletion (Figure 3B, lanes 7 and 8). It has been shown that Mcm4 loaded on chromatin is phosphorylated for initiation of replication at the onset of S-phase (Masai et al, 2006; Sheu and Stillman, 2006, 2010; Randell et al, 2010). We examined whether phosphorylated Mcm4 formed a complex with Psf2 in the absence of Mcm10. Slow-moving forms of Mcm4 preferentially increased in IP fractions at 100 min in both HU-treated and Mcm10-depleted cells (Figure 3B, lanes 5–8), suggesting that phosphorylated Mcm4 was enriched in a complex. The mobility of slow-moving Mcm4 differed slightly between HU-treated and Mcm10-depleted cells (Figure 3B, lanes 6 and 8), probably due to checkpoint-dependent phosphorylations of Mcm4 under HU-arrested conditions (Ishimi et al, 2003, 2004; Bailis et al, 2008). The presence of 700 mM NaAc in IP buffer did not cause significant difference in co-IPed Mcm6 or Cdc45 between Mcm10-depleted and HU-treated cells (Supplementary Figure S5). These results suggest that GINS forms a complex with Cdc45 and Mcm2-7 on chromatin in the absence of Mcm10. Figure 3.Mcm4, Mcm6 and Cdc45 form a complex with Psf2 (GINS) in the absence of Mcm10. (A) Co-precipitation of Mcm6 and Cdc45 with Psf2–Flag. mcm10-off-aid psf2–flag cdc45–myc (Flag+) and mcm10-off-aid cdc45–myc (Flag–) cells were arrested at the G2/M boundary and released in the presence of HU (12 mM). Cell extracts were prepared at 100 min with HU, and proteins were immunoprecipitated with anti-Flag antibody. Co-immunoprecipitated proteins (lanes 3 and 4) were analysed by immunoblotting using anti-Mcm6, anti-Cdc45 and anti-Flag antibodies. The samples used as input (lanes 1 and 2) corresponded to 0.2, 1 and 10% of proteins used for immunoprecipitation of Mcm6, Cdc45 and Psf2, respectively. An asterisk (*) indicates the IgG band. (B) mcm10-off-aid psf2–flag cdc45–myc cells with or without Mcm10 depletion were synchronously released from G2/M block. HU (12 mM) was added to the cells without depletion. Cell extracts were prepared at G2/M release (0 min, lanes 1, 3, 5 and 7) and at 100 min (lanes 2, 4, 6 and 8), and proteins co-immunoprecipitated with Psf2–Flag (lanes 5–8) were analysed by immunoblotting with anti-Mcm6, anti-Mcm4, anti-Cdc45 and anti-Flag antibodies. The samples used as input (lanes 1–4) corresponded to 0.2, 0.1, 1 and 10% of proteins used for immunoprecipitation of Mcm6, Mcm4, Cdc45 and Psf2, respectively. Download figure Download PowerPoint Mcm10 is required for origin DNA unwinding Because components of the CMG complex, which has been shown to exhibit robust DNA unwinding activity in vitro (Moyer et al, 2006; Ilves et al, 2010), bound to replication origins and formed a complex in the absence of Mcm10, we examined whether origin DNA was unwound. The localization of Rpa2, the second largest subunit of the single-stranded DNA-binding protein complex (RPA), was examined by ChIP assay. In HU-treated cells without depletion, Rpa2 was localized at early origins in S-phase (Figure 4A, left, 75 and 90 min). In contrast, Rpa2 was hardly detected at the origins in Mcm10-depleted cells (Figure 4A, right). Because HU causes replication fork arrest resulting in accumulation of RPA, we used unperturbed cells to examine the localization of Rpa2, as well as that of Mcm6 and Psf2 (GINS). At 60–70 min after release from G2/M block, Mcm6, Psf2 and Rpa2 were transiently localized to the origin under normal conditions (Figure 4B–D, left). In Mcm10-depleted cells, origin binding of Rpa2 was greatly decreased, despite accumulation of Mcm6 and Psf2 at the origins (Figure 4B–D, right). These results suggest that Mcm10 is required for the origin DNA unwinding after loading of the CMG components. Since RPA binding is a proxy for ssDNA formation, it could show, for example, that Mcm10 is required for RPA recruitment itself. Figure 4.Mcm10 is required for origin DNA unwinding. (A) mcm10-off-aid cells were arrested at the G2/M boundary with or without Mcm10 depletion and released into the synchronous cell cycle. HU (12 mM) was added to the cells without depletion. Co-immunoprecipitated DNA with Rpa2 was quantified by real-time PCR. IP recoveries (%) are indicated by columns, and error bars show±s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S6. (B–D) mcm10-off-aid cells carrying psf2–flag were arrested at the G2/M boundary with or without Mcm10 depletion and released without HU. DNA co-immunoprecipitated with Mcm6 (B), Psf2–Flag (C) and Rpa2 (D) was quantified at the indicated time points. (E–F) The mcm10-off-aid cdc45–myc strain was arrested at the G2/M boundary, and then released. HU (12 mM) was added to the cells without depletion. At 75 min after G2/M release, localization of Cdc45 (E) and Rpa2 (F) at the indicated distances (kb), on the left (L) and right (R), from the centre of ars2004 was analysed by real-time PCR. Error bars show s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S6. Download figure Download PowerPoint Under conditions where origin unwinding was blocked in the absence of Mcm10, the CMG components would not translocate from replication origins. We examined the distribution of Cdc45 as well as Rpa2 around ars2004. In HU-treated cells without Mcm10 depletion, Cdc45 and Rpa2 were widely distributed up to 20 kb, but not at 30 kb (nonARS1) (Figure 4E and F, left). In sharp contrast, in Mcm10-depleted cells, Cdc45 was localized within a region of 1 kb, but not in distant regions (Figure 4E, right). No significant localization of Rpa2 was detected at any of the positions examined in the absence of Mcm10 (Figure 4F, right). These results suggest that Mcm10 is required for transition of the assembled CMG components into a translocatable helicase complex. Mcm10-dependent origin unwinding is prerequisite for replisome assembly Absence of origin DNA unwinding would impair assembly of the replisome complex. We examined the localization of the catalytic subunits of Pol α (Pol1), Pol δ (Cdc6) and Pol ε (Cdc20), respectively, in the absence of Mcm10. In HU-treated cells without depletion, ars2004 and ars3002, but not nonARS1, were enriched by Pol1-, Cdc6- or Cdc20-IP at 75–90 min, indicating that these factors bound to replication origins (Figure 5A–C, left). In contrast, neither Pol1 nor Cdc6 was localized at the origins under conditions of Mcm10 depletion (Figure 5A and B, right). These results show that Mcm10 is prerequisite for replisome assembly. Although these results are consistent with previous reports, indicating that Mcm10 is required for chromatin binding of Pol α (Ricke and Bielinsky, 2004; Zhu et al, 2007), we did not observe any decrease in the cellular amount of the catalytic subunit in the absence of Mcm10 (Supplementary Figure S8), unlike the reported requirement of Mcm10 for stabilization of Pol α in budding yeast and human cells (Ricke and Bielinsky, 2004, 2006; Chattopadhyay and Bielinsky, 2007). In contrast to Pol1 and Cdc6, Cdc20 accumulated at origins under Mcm10-depleted conditions (Figure 5C, right). Pol ε is essential for assembly of GINS to replication origins before initiation of DNA replication in budding yeast (Muramatsu et al, 2010) and fission yeast (T Handa and H Masukata, unpublished observations). These results suggest that Mcm10 is not required for origin recruitment of Pol ε but prerequisite for recruitment of Pol α and Pol δ, which occurs after origin unwinding. Figure 5.Replisome assembly is dependent on Mcm10. (A–C) mcm10-off-aid cells carrying pol1–flag, cdc6–flag or cdc20–flag were arrested at the G2/M boundary with or without Mcm10 depletion and released into the synchronous cell cycle. HU (12 mM) was added to the cells without depletion. DNA co-immunoprecipitated with Pol1–Flag (Pol α) (A), Cdc6–Flag (Pol δ) (B) and Cdc20–Flag (Pol ε) (C) was measured by real-time PCR. IP recoveries (%) are indicated by columns, and error bars show s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S7. Download figure Download PowerPoint Initiation-specific factors accumulate at replication origins in the absence of Mcm10 Sld3, Dpb11/Cut5 and Sld2/Drc1 are localized at replication origins and required for loading of GINS and Cdc45 to replication origins (Masumoto et al, 2002; Yabuuchi et al, 2006; Tanaka et al, 2007; Zegerman and Diffley, 2007; Fukuura et al, 2011). Because these factors do not translocate with the CMG in the replisome progression complex (Kanemaki and Labib, 2006; Labib, 2010; Taylor et al, 2011), they appear to be released from the CMG at a specific step during or after initiation. To examine whether these factors are released by recruitment of GINS and Cdc45 in the absence of Mcm10, we examined the localization of Sld3, Cut5 and Drc1 by ChIP assay. As a control condition for Cut5 localization, cells were not treated with HU to avoid loading of Cut5 onto the stalled replication forks (Taylor et al, 2011). Sld3 and Cut5 were localized only transiently at origins in cells without HU (Figure 6A and B, left). Drc1 was transiently detected in HU-treated cells without Mcm10 depletion (Figure 6C, left). In contrast, all of these factors were highly accumulated at the replication origins in Mcm10-depleted cells (Figure 6A–C, right). These observations suggest that loading of GINS and Cdc45 does not cause release of Sld3, Cut5 or Drc1, and that they dissociate from origins upon or after recruitment of Mcm10. Figure 6.Sld3, Cut5 and Drc1 accumulate at replication origins in the absence of Mcm10. (A, B) mcm10-off-aid cells carrying sld3–flag and cut5–myc were arrested at the G2/M boundary with or without Mcm10 depletion and released into the synchronous cell cycle. DNA co-immunoprecipitated with Sld3–Flag (A) and Cut5–myc (B) was measured by real-time PCR. IP recoveries (%) are indicated by columns, and error bars show s.d. obtained from triplicate measurements in real-time PCR quantification. The results of biologically independent experiments are presented in Supplementary Figure S9. (C) mcm10-off-aid cells carrying drc1–flag were arrested at the G2/M boundary with or without Mcm10 depletion and released into the synchronous cell cycle. HU (12 mM) was added to the cells without depletion. DNA co-immunoprecipitated with Drc1–Flag was quantified by real-time PCR. Download figure Download PowerPoint To confirm that the above observations are not a side effect of depletion of other protein with Mcm10, we isolated a novel temperature-sensitive mutant, mcm10-5 (Supplementary Figure S10). Using this mutant, we found that Mcm6, Sld3, Cdc45 and Dpb2, the second largest subunit of Pol ε, but not Rpa2, were assembled at replication origins (Supplementary Figure S11), thus supporting the contention that Mcm10 is required for origin unwinding after recruitment of the CMG components. A conserved zinc-finger motif of Mcm10 is essential for origin DNA unwinding To understand the molecular mechanism of origin unwinding that requires Mcm10, we focused on the role of the central domain of Mcm10 that is highly conserved" @default.
• W2165749026 created "2016-06-24" @default.
• W2165749026 creator A5038498098 @default.
• W2165749026 creator A5041891944 @default.
• W2165749026 creator A5043664303 @default.
• W2165749026 creator A5066707889 @default.
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• W2165749026 date "2012-03-20" @default.
• W2165749026 modified "2023-10-18" @default.
• W2165749026 title "Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components" @default.
• W2165749026 cites W1892109385 @default.
• W2165749026 cites W1965813899 @default.
• W2165749026 cites W1967998674 @default.
• W2165749026 cites W1974394081 @default.
• W2165749026 cites W1975001355 @default.
• W2165749026 cites W1986773553 @default.
• W2165749026 cites W1988377515 @default.
• W2165749026 cites W1990299164 @default.
• W2165749026 cites W1992334813 @default.
• W2165749026 cites W1995434980 @default.
• W2165749026 cites W2004536741 @default.
• W2165749026 cites W2006196820 @default.
• W2165749026 cites W2007214324 @default.
• W2165749026 cites W2008685538 @default.
• W2165749026 cites W2012501991 @default.
• W2165749026 cites W2019928852 @default.
• W2165749026 cites W2027290354 @default.
• W2165749026 cites W2027594270 @default.
• W2165749026 cites W2034210492 @default.
• W2165749026 cites W2035405497 @default.
• W2165749026 cites W2038427905 @default.
• W2165749026 cites W2044440192 @default.
• W2165749026 cites W2045437403 @default.
• W2165749026 cites W2049171516 @default.
• W2165749026 cites W2058337905 @default.
• W2165749026 cites W2064154094 @default.
• W2165749026 cites W2065247158 @default.
• W2165749026 cites W2070222783 @default.
• W2165749026 cites W2070538760 @default.
• W2165749026 cites W2071699822 @default.
• W2165749026 cites W2080222318 @default.
• W2165749026 cites W2084518357 @default.
• W2165749026 cites W2089507709 @default.
• W2165749026 cites W2090630813 @default.
• W2165749026 cites W2091570539 @default.
• W2165749026 cites W2092296056 @default.
• W2165749026 cites W2095146606 @default.
• W2165749026 cites W2099487576 @default.
• W2165749026 cites W2100064182 @default.
• W2165749026 cites W2105222529 @default.
• W2165749026 cites W2105413529 @default.
• W2165749026 cites W2106130068 @default.
• W2165749026 cites W2108221620 @default.
• W2165749026 cites W2111275137 @default.
• W2165749026 cites W2113126534 @default.
• W2165749026 cites W2113305379 @default.
• W2165749026 cites W2116004354 @default.
• W2165749026 cites W2119731884 @default.
• W2165749026 cites W2121963528 @default.
• W2165749026 cites W2124221060 @default.
• W2165749026 cites W2126909443 @default.
• W2165749026 cites W2127225607 @default.
• W2165749026 cites W2129585221 @default.
• W2165749026 cites W2130717106 @default.
• W2165749026 cites W2132779860 @default.
• W2165749026 cites W2133657913 @default.
• W2165749026 cites W2134146312 @default.
• W2165749026 cites W2138081166 @default.
• W2165749026 cites W2139535006 @default.
• W2165749026 cites W2142718408 @default.
• W2165749026 cites W2142867416 @default.
• W2165749026 cites W2144021776 @default.
• W2165749026 cites W2148348950 @default.
• W2165749026 cites W2150000259 @default.
• W2165749026 cites W2152115601 @default.
• W2165749026 cites W2164938703 @default.
• W2165749026 cites W2165914686 @default.
• W2165749026 cites W2168241973 @default.
• W2165749026 cites W2236011578 @default.
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