Eukaryotic DNA replication is semi-discontinuous with the leading strand synthesized continuously and lagging strand is synthesized in fragments called Okazaki fragments (1). DNA replication starts at sites called replication origins present along the entire length of the chromosome (2). These sites have Origin of Replication Complex, which recruits different proteins to form replication fork (3).
Helicases are one of those proteins that use energy from ATP hydrolysis to unwind the double stranded DNA to start the replication (4). Mcm2-7 proteins are considered to be a good candidate as helicases, as it has been shown to unwind the short double stranded DNA with 3’-5’ polarity (5). Mcm2-7 proteins have been co-immunoprecipitated with Cdc45 (6), which is an essential factor at the replication fork. GINS has also been shown to be necessary for the function of Cdc45 at the replication fork (7).
Hypothesis: The role of Cdc45 in DNA replication is not well understood although it plays a critical part and has been shown to interact with various DNA replication machinery proteins such as Mcm2-7 and GINS. This study was designed to study the function of Cdc45 in Eukaryotic DNA Replication Summary of the results: 1. Purification and identification of the Cdc45/Mcm2-7/GINS (CMG) complex (Fig 1-2) a. Purification and co-immunoprecipitation of Cdc45 revealed 10 proteins as binding partners (Fig 1A-B). b.
These are identified by mass spectroscopy and immunoblotting to be Mcm2-7 (6 proteins) and GINS complex (S1d5, Psf1, Psf2, and Psf3) proteins (Fig 1A, B). c. Cdc45 does not interact with individual Mcm proteins but only binds as a complex as it existed only in high molecular weight fractions (Fig 1C, Fig 2). d. These proteins form a stable, 11-member, high molecular weight complex. The mass of CMG complex is 700kDa as estimated through migration pattern in a Sepharose column. The calculated mass of one Mcm2-7 hexamer, one GINS tetramer and Cdc45 is 708kDa.
So this CMG complex has only on Mcm2-7 hexamer and one GINS tetramer (Fig 1C, Fig 2). 2. Purification of the CMG complex to homogeneity (Fig 3A) a. The only way to purify intact CMG complex is by using an anti-Cdc45 affinity resin as other methods might disrupt the enzymatic activity of the complex. b. The complex was gently eluted using Cdc45 peptides that are hydrophilic and without secondary structure. c. This method of purification maintained the intrinsic enzymatic activity of the complex.
3. Identification of CMG complex for Helicase activity (Fig 3B – 3E) a. Only the complete 11-member CMG complex had helicase activity when assayed using a 40-bp duplex region with short tail annealed to a single stranded circle. b. Immunodepletion of Psf2 and Mcm5 proteins of the CMG using antisera against those proteins showed reduced helicase activity. This suggests that the activity is associated with the CMG complex and that Psf2 and Mcm5 are components of this complex. c.
The various preparations of CMG complex also showed that the helicase activity is ATP dependent and is saturated at the concentration of 1mM ATP. 4. CMG directionality and processivity (Fig 4) a. Directionality assays using radiolabeled 5’ or 3’ primer with ssDNA in the middle revealed that CMG complex translocates on DNA in a 3’-5’ direction. b. The CMG complex can process substrates with a gapped single strand or 5’ or 3’ overhangs, just like the Mcm4, 6, 7 proteins was determined using a substrate with 5’ 30T tail and gapped single strand. c.
The CMG complex can process many hundreds of base pairs at lower levels of protein compared to the Drosophila Mcm4, 6, 7 sub complex was established by using a population of duplex primer-plasmid regions of various heterogeneous lengths. 5. In Vivo Requirement of Cdc45 and GINS complex (Fig 5) a. RNA interference of Cdc45 or GINS complex proteins in Drosophila Kc tissue culture cells resulted in the accumulation of the cells in G1 and S phase of the cell cycle. This suggests that the cell cycle progression is impaired with the loss of Cdc45 or GINS member proteins.
Conclusion: Cdc45, Mcm2-7 and GINS members form CMG complex, which forms the core of helicase activity for DNA replication. References: 1. Langston, LD. and O’Donnell, M. Mol. Cell 23: 155-160, 2006. 2. Maiorono, D. et al, Curr. Opin. Cell Biol. 18: 130-136, 2006. 3. Bell, SP. and Dutta, A. Annu. Rev. Biochem. 71: 333-374, 2002. 4. von Hippel, PH. And Delagoutte, E. BioEssays 25: 1168-1177, 2003. 5. You, Z. et al, Mol. Cell Biol. 19: 8003-8015, 1999. 6. Masuda, et al, Genes Cells 8: 145-161, 2003. 7. Takayama et al, Genes Dev. 17: 153-1165, 2003.