Presentation on theme: "Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase Stephen E. Moyer, Peter W. Lewis, and Michael."— Presentation transcript:
Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase Stephen E. Moyer, Peter W. Lewis, and Michael R. Botchan
Indirect Evidence points to Mcm2-7 hexamer as part of the replicative helicase 1.Each of the genes is essential and 5 of the 6 have been shown to be required for both DNA Replication initiation and elongation in vivo in S. cerevisiae. 2.Mcm2-7 proteins are required for chromosomal DNA unwinding and that Mcm2, Mcm5, and Mcm7 localize to these sites
Conviction? The purified Mcm 2-7 doesnt have helicase activity only a subcomplex containing Mcm 4,6, and 7 does. But in the presence of the other Mcm(s), the helicase activity is inhibited.
Accomplice? Cdc45 –a pivotal role in G1 to S phase transition –a possible helicase cofactor due to it association with sites of DNA unwinding, genetic interactions with Mcms, and many dna replication proteins –But, no direct link could be established
Purification and Identification of the CMG Complex
Figure 1 Purification Protocol Mcm2-7 and GINS Co-IP with Cdc45 Anti-Cdc45 IP SDS-PAGE 9% 12% Figure 1B
Anti-Cdc45 IP of Superdex-200 fractions Figure 1C Western Blot A 280 Thyroglobulin Catalase BSA Only discrete high-molecular-weight fractions contain CMG complex
Not able to reform CMG complex by mixing of lower molecular-weight fractions Supp. 7 Total protein from Superdex-200 fractions A 280 Thyroglobulin Catalase BSA Western Blot
Figure 1 Summary 1.Mcm2-7 and GINS Co-IP with Cdc45 2.Only discrete high-molecular-weight fractions contain CMG complex Majority of the Cdc45 protein in these extracts exists as a free, low-molecular-weight pool !!!!!! 5 % of total cdc45 and GINs protein and 1% of total Mcms are in the complex 3.Not able to reform CMG complex by mixing of lower molecular-weight fractions Suggest that proteins in complex are modified to permit association
IPs with antibodies against different CMG complex members 10% SDS PAGE Figure 2 Helicase?
The CMG complex is a helicase Figure 3 C Western Blot Phopho- imager Components?
Helicase Activity requires all CMG components and is ATP-dependent Western Helicase Assay Figure 3 Summary
Figure 3 Summary CMG is a helicase –Requires Mcm2-7, GINS, and cdc45 for activity –Helicase activity is ATP-dependent Direction
CMG is as processive as the Mcm 4,6,7 subcomplex Figure 4 Phosphoimager picture of DNA substrates Summary + T7 Polymerase 600bp 400bp 200bp
Figure 4 Summary CMG moves in a 3 to 5 direction CMG is as processive as the Mcm4,6,7 subcomplex FACS
Cdc45 and GINS proteins are required for normal S phase progression Figure 5 Fluorescence # of cells Flow cytometry
Conclusions Cdc4 exists in a stable biochemical unit with the Mcm2-7 and GINS complex Complex is associated with ATP-dependent helicase activity The helicase moves 3 to 5 and is as processive as the Mcm4,6,7 subcomplex Cdc45 and GINS proteins are required for normal S phase progression in metazoans Future Experiment Reconstitution of the complex from recombinant proteins Unanswered Questions How does the CMG complex form? What are the roles of cdc45 and GINS?