DNA replication is required for all dividing cells, and errors in DNA replication can lead to cancer. The replication fork helicase unwinds parental duplex DNA at the front of a replication fork. The replication fork helicase is composed of Cdc45, Mcm2-7, and GINS (CMG complex), and assembly and activation of the CMG complex is central to the process of DNA replication initiation. Sld2, Sld3 and Dpb11 are required for the helicase assembly in budding yeast, and DDK (Dbf4-dependent kinase) and S-CDK (S-phase cyclin dependent kinase) are S phase specific kinases that activate helicase assembly. There are three critical steps to the assembly and activation of the replication fork helicase that are presently unknown: (1) How does the Mcm2-7 double hexamer dissociate to single hexamers in S phase? The Mcm2-7 complex loads as a double hexamer in to encircle double-stranded DNA during G1, but in S phase the Mcm2-7 double hexamer dissociates into single hexamers. It is presently unknown what mediates Mcm2-7 double hexamer disassembly (2) How is Cdc45 recruited to Mcm2-7? Cdc45 binds to Mcm2-7 weakly on its own, and Sld3 or Dpb11 is required to recruit Cdc45 to Mcm2-7 in a process that is dependent upon DDK. However, it is currently not known why DDK is required for Cdc45 recruitment to Mcm2-7, and we will determine this in Aim 2. (3) How is GINS recruited to Mcm2-7? GINS is recruited to Cdc45-Mcm2-7 in S phase, but it is not known how this recruitment occurs. We will determine how GINS is recruited to Cdc45-Mcm2-7 in Aim 3. The Mcm2-7 double hexamer is positioned with the N-terminal domains touching one another. Furthermore, the N-terminal domains of Mcm2-7 are phosphorylated by DDK and S-CDK in vivo.
In Aim 1 we will test the hypothesis that DDK and S-CDK phosphorylation of Mcm2-7 is necessary and/or sufficient for Mcm2-7 double hexamer disassembly.
In Aim 2 we will test different hypotheses to explain how DDK activates Cdc45 recruitment to Mcm2-7.
In Aim 3, we will test the prevailing view in the field, that Dpb11 directly recruits GINS to Mcm2-7. We will also test an alternative hypothesis, that Dpb11 competes with GINS for binding to Mcm2-7 in G1, but in S phase, as single-stranded DNA is extruded from the central channel of Mcm2-7, Dpb11 binds to ssDNA preferentially, allowing GINS to bind Mcm2-7. Completion of these three aims will allow for a functional and mechanistic understanding of how the eukaryotic replication fork helicase is assembled and activated in eukaryotes. Compounds that inhibit cell cycle kinases are promising for the treatment of cancer, and therefore DDK and S-CDK are logical targets for cancer treatment. Mcm proteins are currently used as prognostic markers for cancer. Thus, further understanding of how Mcm, DDK, and S-CDK function may lead to improvements in cancer diagnostics and therapeutics.
Mcm2-7 proteins are useful as markers in the prognosis and diagnosis of cancer. Furthermore, the cell cycle kinases DDK and S-CDK are current targets for chemotherapy. Further understanding of DNA replication initiation will lead to improvements in the diagnosis and treatment of cancer.
