isolation of the cdc45/mcm2-7/gins (cmg) complex, a candidate for the eukaryotic dna replication...

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