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A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication

2004; Springer Nature; Volume: 23; Issue: 18 Linguagem: Inglês

10.1038/sj.emboj.7600369

1460-2075

Marcin Pacek, Johannes C. Walter,

Genomics and Chromatin Dynamics

Article26 August 2004free access A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication Marcin Pacek Marcin Pacek Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Johannes C Walter Corresponding Author Johannes C Walter Search for more papers by this author Marcin Pacek Marcin Pacek Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Johannes C Walter Corresponding Author Johannes C Walter Search for more papers by this author Author Information Marcin Pacek1 and Johannes C Walter 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA *Corresponding author. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, BCMP, C2-Room 322, 240 Longwood Avenue, Boston, MA 02115, USA. Tel.: +1 617 432 4799; Fax: +1 617 738 0516; E-mail: [email protected] The EMBO Journal (2004)23:3667-3676https://doi.org/10.1038/sj.emboj.7600369 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In vertebrates, MCM2–7 and Cdc45 are required for DNA replication initiation, but it is unknown whether they are also required for elongation, as in yeast. Moreover, although MCM2–7 is a prime candidate for the eukaryotic replicative DNA helicase, a demonstration that MCM2–7 unwinds DNA during replication is lacking. Here, we use Xenopus egg extracts to investigate the roles of MCM7 and Cdc45 in DNA replication. A fragment of the retinoblastoma protein, Rb1−400, was used to neutralize MCM7, and antibodies were used to neutralize Cdc45. When added immediately after origin unwinding, or after significant DNA synthesis, both inhibitors blocked further DNA replication, indicating that MCM7 and Cdc45 are required throughout replication elongation in vertebrates. We next exploited the fact that inhibition of DNA polymerase by aphidicolin causes extensive chromosome unwinding, likely due to uncoupling of the replicative DNA helicase. Strikingly, Rb1−400 and Cdc45 antibodies both abolished unwinding by the uncoupled helicase. These results provide new support for the model that MCM2–7 is the replicative DNA helicase, and they indicate that Cdc45 functions as a helicase co-factor. Introduction Vertebrate cells contain a vast amount of DNA that is faithfully replicated during every mitotic cell division (for reviews, see Waga and Stillman, 1998; Bell and Dutta, 2002; Mendez and Stillman, 2003). The first step in DNA replication, pre-RC assembly, occurs at thousands of origins during the G1 phase of the cell cycle when a six-subunit origin recognition complex (ORC) bound to origins recruits the initiation factors Cdc6, Cdt1, and MCM2–7. Once MCM2–7 is loaded, ORC becomes dispensable, and the MCM2–7 complex serves as the platform on which further initiation events take place (Hua and Newport, 1998; Rowles et al, 1999; Shimada et al, 2002). In S phase, pre-RCs are acted upon by two protein kinases and a multitude of replication initiation factors, which together are required for origin unwinding. In metazoans, MCM10 and the protein kinase Cdc7/Dbf4 are the first factors to load onto pre-RCs, and their loading is MCM2–7-dependent (Jares and Blow, 2000; Walter, 2000; Wohlschlegel et al, 2002). MCM10 and Cdc7 enable the loading of several additional factors such as GINS and Cdc45, whose binding is also dependent on Cdk2/Cyclin E. The binding of Cdc45 and GINS to pre-RCs is interdependent and converts this structure into a pre-Initiation Complex (or pre-IC) (Mimura and Takisawa, 1998; Zou and Stillman, 1998; 2000; Jares and Blow, 2000; Walter, 2000; Kubota et al, 2003; Takayama et al, 2003). Formation of the pre-IC is the last known event that occurs before origin unwinding, which is accompanied by chromatin loading of the single-stranded DNA-binding protein RPA (Tanaka and Nasmyth, 1998; Mimura et al, 2000; Walter and Newport, 2000). Once the origin has been sufficiently unwound, DNA polymerase α loads and synthesizes an RNA primer, which it then extends to form a short DNA primer. The presence of a DNA primer allows loading of the processivity factor PCNA by the RFC complex, followed by pol δ. The final stage of DNA replication, elongation, involves the coordinated synthesis of nascent strands. Studies in yeast clearly show that, in addition to DNA polymerases, elongation also requires MCM2-7, Cdc45, and GINS (Labib et al, 2000; Tercero et al, 2000; Kanemaki et al, 2003), all of which localize to replication forks (Aparicio et al, 1997; Kanemaki et al, 2003; Takayama et al, 2003). It is unknown whether MCM2–7, Cdc45, or GINS are also required for elongation in metazoans. Given the results in yeast, it is surprising that immunofluorescence studies in vertebrate cells failed to detect colocalization of MCM2–7 with sites of ongoing DNA replication (Todorov et al, 1994; Krude et al, 1996; Romanowski et al, 1996). The requirement for MCM2–7 in elongation in yeast is consistent with it being the eukaryotic replicative DNA helicase (Labib and Diffley, 2001). Like the replicative DNA helicases DnaB and Large T antigen, all six MCM subunits are members of the AAA+ family of ATPases, and the MCM2–7 complex adopts a ring-like structure (Chong et al, 2000; Fletcher et al, 2003). In yeast and mammals, a purified MCM4/6/7 subcomplex exhibits helicase activity in oligo-nucleotide displacement assays (You et al, 1999; Lee and Hurwitz, 2000; Kaplan et al, 2003). However, MCM2–7 is inactive as a helicase, and the purified MCM4/6/7 complex is inhibited by MCM2 and MCM3/5 (Ishimi et al, 1998; Lee and Hurwitz, 2000), which is surprising given the requirement for all six MCM subunits in fork progression in vivo (Labib et al, 2000). These observations have led to models in which MCM4/6/7 is the motor that unwinds DNA, whereas the other subunits serve regulatory functions. A definitive demonstration that MCM2–7 is the eukaryotic replicative DNA helicase will require biochemical reconstitution of DNA replication or a demonstration that MCM2–7 performs helicase activity at the replication fork (Labib and Diffley, 2001). Unlike the MCM2–7 complex, Cdc45 contains no known sequence motifs, and its molecular mechanism is unclear. Interestingly, Cdc45 is found in a complex with MCM2–7 on chromatin in yeast and in Xenopus egg extracts (Zou and Stillman, 1998; Mimura et al, 2000), suggesting that it might regulate the activity of MCM2–7. To study chromosomal DNA replication, we use a soluble cell-free system derived from Xenopus eggs. Sperm chromatin or plasmid DNA is first incubated in a high speed supernatant of egg cytoplasm (HSS), leading to pre-RC assembly. Subsequently, a highly concentrated nucleoplasmic extract (NPE) prepared from synthetic nuclei is added, which supplies Cdk2/Cyclin E and Cdc7/Dbf4, as well as other activities (Walter, 2000; Wohlschlegel et al, 2002; Prokhorova et al, 2003) and a complete round of DNA replication ensues. Using this system, origin unwinding can be detected on plasmids via negative supercoiling, or on sperm via RPA loading (Walter and Newport, 2000). Interestingly, in the presence of aphidicolin, which inhibits replicative DNA polymerases, the degree of negative supercoiling and RPA loading are dramatically enhanced, indicating a high degree of DNA unwinding, or ‘hyperunwinding’ (Walter, 2000; Walter and Newport, 2000). Since hyperunwinding depends on prior initiation of DNA replication, and is rapid and extensive, it likely reflects the action of the replicative DNA helicase after it has become uncoupled from the stalled replication complex. Thus, aphidicolin-induced helicase uncoupling represents a potentially powerful assay to study the eukaryotic replicative DNA helicase in the context of replication-competent chromatin. We wanted to determine whether Cdc45 and the MCM complex are required after pre-IC formation in Xenopus egg extracts. To inactivate the chromatin-bound MCM complex after pre-ICs had formed, we used the N-terminal 400 amino acids of Rb (Rb1–400), a domain that was previously shown to interact with MCM7 and thereby inhibit DNA replication in Xenopus egg extracts at an unknown step (Sterner et al, 1998). When we added Rb1–400 to DNA replication complexes synchronized immediately after origin unwinding or during elongation, it completely inhibited further DNA replication, indicating a role for MCM7 in elongation. Moreover, at both of these stages, Rb1–400 inhibited the activity of the uncoupled DNA helicase in the presence of aphidicolin. These experiments show a direct role for MCM7 in DNA unwinding in the context of replication-competent chromatin, and thus provide new support for the idea that the MCM complex is the replicative DNA helicase. To determine whether Cdc45 is required for elongation, we inactivated chromatin-bound Cdc45 using antibodies. Cdc45 antibodies inhibited DNA replication and DNA unwinding by the uncoupled DNA helicase when added to replication complexes synchronized immediately after origin unwinding, or during elongation. The data show that Cdc45 is required for chromosome unwinding during elongation, and they are consistent with a model in which Cdc45 stimulates the helicase activity of the MCM2–7 complex. Results Rb1–400 inhibits origin unwinding when added after pre-RC formation To examine what steps of DNA replication after pre-IC formation are dependent on the MCM complex, we sought to inactivate the chromatin-bound complex at progressively later stages of DNA replication. To this end, we took advantage of the previous observation that the retinoblastoma gene product, Rb, binds to MCM7 (Sterner et al, 1998). Using two-hybrid assays and co-immunoprecipitation of mammalian cell extracts, Rb1–400 was found to interact with the C-terminal region of MCM7. Moreover, when Rb1–400 was added to nuclear-assembly egg extracts before sperm chromatin, DNA replication was blocked. The inhibition was reversed when Rb1–400 was pre-incubated with an MCM7 peptide, arguing that inhibition was the result of Rb1–400 binding to the endogenous MCM7 protein (Sterner et al, 1998). It was not determined which step in DNA replication is blocked by Rb1–400. We examined the effects of Rb1–400 on DNA replication in the nucleus-free system (see Introduction). Initially, Rb1–400 was added to HSS before sperm chromatin. Upon addition of NPE, DNA replication was blocked, and the inhibition was relieved by the MCM7 peptide (data not shown). Further analysis showed that, in the presence of Rb1–400, the MCM complex and Cdc45 failed to load onto chromatin (data not shown). These results show that, when added before sperm chromatin, Rb1–400 can block pre-RC assembly, and they confirm the previous observation that Rb1–400 inhibits DNA replication in Xenopus egg extracts (Sterner et al, 1998). We next sought to determine what happens when Rb1–400 is added after MCM2–7 complexes have loaded onto chromatin. Sperm chromatin was incubated in HSS to assemble pre-RCs. Subsequently, Rb1–400 was added, followed by NPE. Under these conditions, Rb1–400 inhibited DNA replication five-fold, but when Rb1–400 was pre-incubated with MCM7 peptide, inhibition was relieved (Figure 1A, bar graph). To determine at what stage DNA replication was blocked, chromatin was isolated from NPE containing aphidicolin (NPEaph) to examine which factors loaded in the presence of Rb1–400. As discussed in the Introduction, the hyperloading of RPA in the presence of aphidicolin likely reflects the action of the replicative DNA helicase after it has become uncoupled from the stalled replication complex. In the presence of Rb1–400, MCM7, ORC2, and Cdc45 binding was unaffected, but RPA hyperloading was reduced (Figure 1A, upper panel, compare lanes 1 and 2), and the effect was reversed when Rb1–400 was preincubated with MCM7 peptide (Figure 1A, lane 3). Importantly, Rb1–400 did not affect the binding of RPA to an immobilized single-stranded DNA oligonucleotide in extracts (data not shown), indicating that the inhibition of RPA loading by Rb1−400 was due to an effect on chromosome unwinding. Figure 1.Rb1–400 inhibits origin unwinding after pre-RC formation. (A) Chromatin-loading assay. Sperm chromatin (10 000/μl) was incubated with 2 μl of HSS for 30 min and then supplemented with buffer (lane 1), 800 ng Rb1–400 (lane 2), or 800 ng Rb1–400 preincubated with 1.6 μg MBP-MCM7 peptide fusion (lane 3). After 30 min, 4 μl NPE was added, which contained aphidicolin (50 μg/ml) and buffer (lane 1), 400 ng Rb1–400 (lane 2), or 400 ng Rb1–400 and 800 ng MCM7 peptide (lane 3). After 45 min, the chromatin was purified, and bound proteins were analyzed by Western blot analysis using antibodies against RPA (lower panel), and a mixture of antibodies against MCM7, ORC2, and Cdc45 (upper panel). To measure DNA replication, the same reaction was carried out using NPE lacking aphidicolin but containing [α-32P]dATP (lower panel). (B) DNA topology assay. Same as panel A, except that pBS (40 ng/μl) was used as the DNA template. Lane 2 shows the effect of p27Kip addition. After 30 min incubation with NPE, the DNA was extracted, separated on a chloroquine agarose gel, and stained (top panel). To measure DNA replication, the same reaction was carried out using NPE lacking aphidicolin but containing [α-32P]dATP (lower panel). (C) Sperm chromatin was incubated with HSS supplemented with control buffer (lanes 1 and 3), or 500 nM geminin (lane 2) for 30 min. Subsequently, buffer (lane 1), 800 ng Rb1–400 (lane 2), or 800 ng Rb1–400 preincubated with 1.6 μg MCM7 peptide (lane 3) was added. After further 30 min, chromatin-bound proteins were analyzed with antibodies against MCM4, Rb, or ORC2. Download figure Download PowerPoint To confirm by another method that Rb1–400 inhibits origin unwinding when added to assembled pre-RCs, we used a DNA topology assay (Walter and Newport, 2000). DNA replication is carried out using a circular plasmid, such as pBluescript (pBS), as the DNA template. Upon initiation of DNA replication, the plasmid becomes transiently underwound, generating a negatively supercoiled species that is readily detected by its rapid mobility during electrophoresis (U-form DNA). Addition of aphidicolin to the NPE traps all the DNA in the U form, and each plasmid undergoes much more extensive unwinding (‘hyperunwinding’) than in the absence of aphidicolin. Hyperunwinding in the presence of aphidicolin is sensitive to the Cdk2 inhibitor p27Kip (Walter and Newport, 2000; Figure 1B, upper panel, compare lanes 1 and 2), indicating that it reflects uncoupling of the replicative DNA helicase after initiation. To determine whether Rb1–400 affected origin unwinding when added after pre-RC formation, pBS was incubated in HSS to form pre-RCs. Subsequently, Rb1–400 was added, followed by NPE containing aphidicolin. Rb1–400 significantly reduced the generation of U-form DNA (Figure 1B, lanes 1 and 3), and inhibition was largely alleviated when Rb1–400 was pre-incubated with MCM7 peptide (Figure 1B, lane 4). Consistent with the inhibition of origin unwinding, Rb1–400 also blocked DNA replication of pBS when it was added after pre-RC formation (Figure 1B, bar graph). We wanted to verify that Rb1–400 targeted MCM7 on chromatin. Sperm chromatin was incubated with HSS to load the MCM complex, followed by addition of Rb1–400. Subsequently, the chromatin was isolated and blotted for Rb. Figure 1C (lane 1) shows that Rb1–400 cosedimented with the chromatin. Importantly, in the presence of geminin, which blocks MCM2–7 chromatin loading by targeting Cdt1 (Wohlschlegel et al, 2000; Tada et al, 2001), Rb1–400 loading was reduced (Figure 1C, lane 2). When Rb1–400 was pre-incubated with MCM7 peptide, Rb1–400 binding was also severely reduced (Figure 1C, lane 3). These results argue that Rb1–400 exerts its inhibitory effects in DNA replication and origin unwinding by binding to MCM7 on chromatin. Together, the data in this section indicate that MCM7 is required for origin unwinding independently of its role in pre-IC formation. Rb1–400 inhibits chromosome unwinding by a previously activated helicase The origin-unwinding defect seen in Figure 1 could be explained in two ways. First, Rb1–400 might block an unknown step after pre-IC formation that is required to activate the replicative DNA helicase, or Rb1–400 might block the fully activated helicase itself. To distinguish between these possibilities, we added Rb1–400 to chromatin containing a previously activated helicase. To this end, sperm chromatin was incubated with HSS, followed by NPE containing actinomycin D (NPEactD). ActD abolishes DNA replication in Xenopus egg extracts (Michael et al, 2000; our data not shown), likely due to its inhibition of the RNA priming activity of DNA pol α (Grosse and Krauss, 1985). Consistent with such a mechanism, actD does not affect chromatin loading of Cdc45 (Edwards et al, 2002) or DNA polymerase α (data not shown). Importantly, in the presence of actD, RPA was also loaded onto chromatin, and the loading was Cdk2-dependent, demonstrating that it required initiation (Figure 2A, compare lanes 5 and 6). The amount of RPA loaded in the presence of actD was similar to the peak level seen on chromatin during an unperturbed S phase (Figure 2A, compare lanes 2 and 5), indicating that unwinding in NPEactD was significant, but it was far less than what is observed during hyperunwinding in aphidicolin extract (Figure 2A, compare lanes 5 and 7). The reason why actD does not support hyperunwinding is unclear. One possibility is that its intercalating activity (Kamitori and Takusagawa, 1992) prevents the ability of the helicase to travel more than a short distance along DNA. Whatever the precise mechanism of actD, the data show that it arrests DNA replication after a Cdk2-dependent helicase has been allowed to unwind a limited amount of DNA (Figure 2B, top). Figure 2.Rb1–400 protein inhibits DNA replication and chromosome unwinding after initiation. (A) RPA loading in the presence of actinomycin D is Cdk2-dependent. Sperm chromatin was incubated with HSS, followed by unsupplemented NPE (lanes 1–4), or NPE containing 10 μM actinomycin D (lanes 5), actinomycin D and p27Kip (lane 6), or aphidicolin (lane 7). At the indicated times, chromatin was isolated and blotted for MCM7, ORC2, Cdc45, and RPA. (B) Model for the actinomycin D and aphidicolin arrest points. (C) Sperm chromatin was incubated with HSS, followed by NPEactD, and isolated (lane 1), or isolated and then incubated with buffer (lane 2), 800 ng Rb1−400 (lane 3), or 800 ng Rb1−400 preincubated with 1.6 μg MCM7 peptide. Isolation leads to permanent immobilization of the sperm on the tube. After 30 min, the supernatant was replaced with 5 μl fresh NPEaph containing buffer (lane 2), 400 ng Rb1−400 (lane 3), or 400 ng Rb1−400 preincubated with 800 ng MCM7 peptide. After 45 min, chromatin was washed and blotted for MCM7, ORC2, Cdc45, and RPA34 (upper panel). Identical reactions were carried out in which the second incubation with NPE lacked aphidicolin but contained [α-32P]dATP to measure DNA replication (bar graph). Download figure Download PowerPoint When chromatin containing the activated helicase was transferred from NPEactD to fresh NPE, DNA replication was efficient, indicating that a replication complex stalled in actD can resume DNA synthesis (Figure 2C, bar graph, column 2). When the chromatin incubated in NPEactD was first exposed to Rb1–400 before transfer to fresh NPE, DNA replication was severely inhibited, but this inhibition was not observed when Rb1–400 was first pre-incubated with MCM7 peptide (Figure 2C, bar graph, compare columns 3 and 4). Therefore, MCM7 is still required for DNA replication after helicase activation. The experiment was repeated, but chromatin was transferred from NPEactD into NPEaph, and chromatin association of RPA was measured. There was a large increase in RPA binding when chromatin was transferred from NPEactD into NPEaph (Figure 2C, upper panel, compare lanes 1 and 2). This increase was dependent on the presence of aphidicolin because it did not occur when chromatin was transferred into fresh NPEactD (Supplementary Figure S1). Moreover, it did not involve new initiation events because it still occurred when chromatin was transferred into NPEaph that also contained the Cdk2 inhibitor, p27Kip (Supplementary Figure S1). Therefore, a helicase arrested in the presence of actD can subsequently become uncoupled from the replication fork and carry out hyperunwinding in the presence of aphidicolin (illustrated in Figure 2B). When chromatin incubated in NPEactD was exposed to Rb1–400 before transfer into NPEaph, RPA hyperloading was strongly reduced, and the effect was reversed by MCM7 peptide (Figure 2C, upper panel, lanes 3 and 4). The data indicate that, even after the replicative DNA helicase has been activated, DNA replication still requires MCM7 due to a direct involvement of this protein in chromosome unwinding. Rb1−400 inhibits DNA replication and chromosome unwinding during replication elongation Although there is origin unwinding of DNA templates incubated in NPEactD(Figure 2A), there is no DNA synthesis (Michael et al, 2000; our data not shown). Therefore, replication complexes assembled in these extracts have not entered the elongation phase of DNA replication. To address whether Rb1–400 inhibits DNA replication of chromatin engaged in elongation, we synchronized replication complexes in elongation mode by lowering the reaction temperature from 22 to 19°C, which caused S phase to be extended by at least 30 min (Figure 3A). To determine the synchrony of origin firing at 19°C, we added p27Kip 20 min after the addition of NPE and found that it had no effect on the kinetics or efficiency of DNA replication (Figure 3B, compare circles and triangles), whereas when p27Kip was added at the same time as NPE DNA replication was completely blocked (Figure 3B, squares). We know that Cdk2 remained inactive throughout the experiment because a fresh, licensed template added after p27Kip did not undergo DNA replication (data not shown). The data indicate that, at 19°C, all origins fire within 20 min of NPE addition, and the time following Cdk2 addition comprises the elongation phase of DNA replication. Figure 3.Rb1–400 inhibits DNA replication and chromosome unwinding during elongation. (A) DNA replication kinetics at 22 and 19°C. Sperm chromatin was pre-incubated with HSS, NPE was added, reactions were transferred to 22°C (squares) or 19°C (circles), and DNA replication was measured. (B) All origins fire within 20 min of NPE addition at 19°C. Sperm chromatin was incubated with HSS, followed by NPE addition and transfer to 19°C. Buffer (circles) or p27Kip was added 0 min (squares) and 20 min (triangles) after NPE. (C) Rb1–400 inhibits the elongation complex. Sperm chromatin was incubated with HSS, followed by addition of NPE and transfer to 19°C. After 25 min, p27Kip was added, and after a further 15 min chromatin was isolated (lane 1). Chromatin was exposed to buffer (lane 2), Rb1–400 (lane 3), or Rb1–400/MCM7 (lane 4). Finally, fresh NPE containing aphidicolin, p27Kip, and buffer (lane 2), Rb1–400 (lane 3), or Rb1–400/MCM7 peptide (lane 4) was added to measure chromatin binding (lower panel). To measure DNA replication (bar graph), aphidicolin was omitted and both NPEs contained [α-32P]dATP. Download figure Download PowerPoint We used the low-temperature synchronization to address whether Rb1–400 inhibits DNA replication of elongating complexes. Pre-RCs were assembled in HSS and allowed to initiate DNA replication in NPE at 19°C. After 25 min, p27Kip was added to inhibit additional initiation events, and after a further 15 min, the chromatin was isolated. At this stage, 30% of the input DNA had replicated (Figure 3C, bar graph, column 1). Individual aliquots of chromatin were then mixed with buffer, Rb1–400, or Rb1–400 and MCM7 peptide, followed by fresh NPE, which was supplemented with p27Kip to prevent new initiation events. In the buffer control, addition of NPEKip allowed a further 20% DNA replication (Figure 3C, column 2). It should be noted that the relatively low efficiency of DNA replication observed after chromatin isolation is due to nonspecific inactivation of chromatin (Walter et al, 1998), but it does not affect our conclusions. In contrast, only ∼1% further replication was observed in the presence of Rb1–400, and 16% additional DNA replication was seen in the presence of Rb1–400 and MCM7 peptide (Figure 3C, columns 3 and 4). These results indicate that MCM7 is required for replication elongation. To determine the effects of Rb1–400 on chromosome unwinding by elongating complexes, chromatin was treated as described above, but it was incubated with NPEKip+aph in the final step to measure RPA hyperloading. As seen in Figure 3C (lower panel), transfer of elongating complexes into NPEKip+aph caused hyperloading of RPA, indicating that the helicase can become uncoupled from elongating complexes (compare lanes 1 and 2). As with DNA replication, Rb1−400 blocked RPA hyperloading, and the effect was reversed by MCM7 peptide (Figure 3C, lanes 3 and 4). The data show that Rb1–400 inhibits DNA replication and chromosome unwinding of elongating replication complexes, consistent with the idea that MCM7 is directly required for chromosome unwinding in these complexes. In Figure 3C, it was conceivable that Rb inhibited DNA replication of the isolated chromatin due to a requirement for MCM7 in restarting the replication fork upon transfer to fresh NPE. However, this scenario is unlikely, since Rb (but not Rb pre-incubated with MCM7 peptide) inhibited DNA replication when added to elongating complexes that had not been isolated from the extract (Supplementary Figure S2). Previously, it was shown that multiple MCM2–7 complexes bind to chromatin in a distributed pattern in Xenopus egg extracts (Edwards et al, 2002; Harvey and Newport, 2003). Therefore, it was conceivable that Rb1–400 might block DNA replication and chromosome unwinding by associating with multiple MCM2–7 complexes, thereby creating a chromatin structure that cannot be traversed by the replication complex. If this model were correct, chromatin with a lower density of MCM2–7 complexes should be impervious to inhibition by Rb1–400. To generate such chromatin, we incubated sperm in HSS that was diluted 10-fold with buffer. Under these conditions, the amount of chromatin-bound MCM2–7 complexes was severely reduced compared to undiluted HSS (Figure 4A, compare lanes 1 and 3), but the residual MCM2–7 loading was still geminin-sensitive (Figure 4A, lane 4). To test whether Rb1–400 still inhibits DNA replication of chromatin containing low levels of MCM2–7, we performed a low-temperature synchronization in which we used either undiluted or 10-fold diluted HSS. Figure 4B shows that DNA replication of chromatin assembled in dilute HSS was ∼50% of control (compare columns 1 and 2), consistent with previous findings that MCM complexes are present on chromatin in a large functional excess (Mahbubani et al, 1997; Edwards et al, 2002), and replication was still completely geminin-sensitive (compare columns 2 and 4). Importantly, despite the low level of MCM2–7 bound, Rb1–400 inhibited DNA replication of chromatin assembled in dilute HSS to the same extent as control chromatin (compare columns 1 and 5 with 2 and 6), and in each case MCM7 peptide rescued DNA replication (columns 7 and 8). This result argues against the idea that inhibition by Rb1–400 results from its association with multiple MCM complexes to generate a repressive chromatin structure. Figure 4.Rb1–400 inhibits DNA replication of chromatin containing low levels of MCM2–7. (A) Effect of HSS dilution on MCM2–7 loading. Sperm chromatin was incubated with HSS (lanes 1 and 2), or HSS that was diluted 10-fold with ELB (lanes 3 and 4), in the presence (lanes 2 and 4) and absence (lanes 1 and 3) of geminin. After 30 min, the chromatin was isolated and blotted for MCM7 and ORC2. (B) Effect of Rb1−400 on chromatin containing low levels of MCM2–7. Sperm chromatin was incubated with HSS (1 ×) or 10 times diluted HSS (0.1 ×), some of which contained 500 nM geminin (lanes 3 and 4). Subsequently, NPE was added at 19°C, and after 25 min p27Kip was added. After a further 15 min, the chromatin was isolated and exposed to buffer, Rb1−400, or Rb1−400/MCM7 peptide. Fresh NPE containing buffer, Rb1−400 or Rb1−400/MCM7 peptide was added, and DNA replication was measured after 45 min. Download figure Download PowerPoint Cdc45 is required for DNA replication and chromosome unwinding after initiation In Cdc45-depleted egg extracts, pre-IC formation is defective, and no origin unwinding is detected (Mimura and Takisawa, 1998; Mimura et al, 2000; Walter and Newport, 2000). To determine whether Cdc45 is required after pre-IC formation, we used a high-titer polyclonal antibody raised against the Cdc45 protein (Walter and Newport, 2000). When added to egg extracts before sperm chromatin, this antibody blocked Cdc45 loading and inhibited DNA replication (data not shown). To determine whether Cdc45 is still required after helicase activation, we incubated sperm chromatin in NPEactD, which allows Cdc45 loading and limited chromosome unwinding (Figure 2B). After isolation, the chromatin was incubated with buffer or purified anti-Cdc45 IgG. The chromatin was washed and supplemented with fresh NPE lacking