After 8 days of RNAi treatment, cells were harvested; FACS profiles are shown

After 8 days of RNAi treatment, cells were harvested; FACS profiles are shown. Mcm5, and Mcm7 localize to sites of DNA unwinding on a plasmid (4C6). The primary structure of the proteins places each in the helicase supergroup of the AAA+ family, and archaeal forms do show robust helicase function (7C9). Curiously, the purified Mcm2C7 complex has not displayed helicase activity egg extracts, Cdc45 associates with sites of DNA unwinding on a plasmid (6), and interfering antibodies directed at Cdc45 abolish chromosomal unwinding (4). There is a conserved genetic interaction between Cdc45 and the Mcms; specific mutations in these genes can either suppress or show synthetic lethality with the other (19C21). By coimmunoprecipitation/immunoblot experiments, Cdc45 has been shown to associate with a variety of other DNA replication proteins, including DNA polymerase (22, 23), various Mcm2C7 proteins (16, 23, 24), Mcm10 (25), Orc2 (26), Sld3 (27), and Sld5 (28). Many of these interactions may be important but transient or indirect and may obscure a core function or complex within which Cdc45 works. Results Purification and Identification of the Cdc45/Mcm2C7/GINS (CMG) Complex. A protocol to purify a high-molecular-weight complex containing the Cdc45 protein from embryo extracts is outlined in Fig. 1(see and see SIX3 also Table 1 and and Fig. 6, which is published as supporting information on the PNAS web site, Cdc45 coimmunoprecipitates with 10 other major proteins. The identity of the copurifying proteins was determined by two methods: (homologs of the four members of the GINS complex (Sld5, Psf1, Psf2, and Psf3, respectively) 3-Hydroxyisovaleric acid (Fig. 1(see Table 2, which is published as supporting information on the PNAS web site). The only DNA replication proteins identified by this method were also Cdc45, Mcm2C7, and GINS. Sld5, the founding member of the GINS complex, was first uncovered in a synthetic lethal screen with Dpb11 (29). Dpb11 was itself genetically isolated in a suppressor screen for a mutant in a subunit of DNA polymerase (30). The Psf genes are each partners of Sld5 and are required for DNA synthesis in yeast and in cell-free DNA replication extracts (28, 29). The GINS proteins form a tetramer, and it was recently shown that Sld5 localizes to sites of DNA unwinding on a plasmid in egg extracts (6), suggesting that all four GINS proteins may be present in a DNA helicase complex. Examination of the Superdex-200 column fractions from Fig. 1shows that the majority of the Cdc45 protein in these extracts exists as a free, low-molecular-weight pool (see Fig. 1and Fig. 7, which is published as supporting information on the PNAS web site). However, only discrete high-molecular-weight fractions contain a complex that shows an interaction among Cdc45, the GINS members, and the 3-Hydroxyisovaleric acid Mcm proteins. Fig. 3-Hydroxyisovaleric acid 1shows an immunoblot of the material eluted from an anti-Cdc45 IP from each Superdex-200 chromatography fraction. The size of the complex as estimated from the elution position on the column closely matches the sum of the individual components (see expressing a functional, FLAG-tagged version of Mcm6 (32). A single-step affinity purification of FLAG-Mcm6 from these flies captures mainly FLAG-Mcm6 in complex with Mcm2, Mcm4, and Mcm7 (Fig. 2shows a Deep Purple stain of the material eluted from the anti-Psf2 IP. Cdc45 and the complete Mcm2C7 complex coimmunoprecipitated with the GINS complex, and immunoblots of the material eluted from each IP (see Fig. 8, which is published as supporting information on the PNAS web site) confirmed the tight association of each of the 11 members of this complex. Based on these multiple lines of evidence, we conclude that Cdc45, Mcm2C7, and GINS form a stable, high-molecular-weight biochemical unit, which we refer to as the CMG complex. An immediate question was whether the complex contained an active form of the hypothetical Mcm2C7 helicase. Open in a separate window Fig. 2. IPs with antibodies against different CMG complex members. In this figure, the proteins were identified by immunoblot and was used to obtain 10 l of the material in and with varying concentrations of ATP. Identification of CMG-Associated Helicase Activity. We used the anti-Cdc45 peptide B-specific antibodies to purify the CMG complex according to the procedure outlined in Fig. 3and asked whether the purified.