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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 doesn’t 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
A28
0
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
A28
0Thyroglobulin
Catalase
BSA
WesternBlot
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
WesternBlot
Phopho-imager
Components?
Helicase Activity requires all CMG components and is ATP-dependent
Western Helicase Assay
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 (helicase) moves 3’ to 5’
Figure 4
Radiolabel
Radiolabel
Phospho-ImagerPicture
Processive
CMG is as processive as the Mcm 4,6,7 subcomplex
Figure 4
Phosphoimager picture of DNA substrates
Summary
+T7 Polymerase
600bp400bp200bp
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 5Fluorescence
# of
cel
ls
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 ExperimentReconstitution of the complex from recombinant proteins
Unanswered Questions
•How does the CMG complex form?•What are the roles of cdc45 and GINS?
R. Sears, Con 664 lecture