Doctor of Philosophy
Dr. David Edgell
The transfer of genetic material from one cell generation to the next requires precise genome duplication. Aberrant DNA replication can lead to genomic instability and contribute to diseases arising from an unregulated cell cycle, such as cancer. Replicative DNA polymerases require a single-stranded (ssDNA) template from which to produce newly synthesized DNA. In eukaryotes, ssDNA is generated by the heterohexameric minichromosome maintenance 2 through 7 (Mcm2-7) replicative helicase that unwinds duplex DNA. Strict temporal separation of helicase loading and activation at multiple replication origins ensures once per cell cycle replication. The processes involved in activating Mcm2-7 to unwind DNA during S phase are poorly understood. Through in vivo and in vitro analyses, the current study examines the factors involved in modulating S. cerevisiae Mcm2-7 activity.
Mec1, a member of the PIKK (phosphoinositide three-kinase-related kinase) family of proteins, is involved in the response to replicative stress and DNA damage. It also plays a role during an unperturbed cell cycle and is required to phosphorylate Mcm2-7 prior to helicase activation. We characterized alleles of S. cerevisiae mec1 that alter the conserved FATC domain. Mutants of Mec1 resulted in temperature sensitive growth, sensitivity to hydroxyurea and reduced kinase activity in vitro. These mutants were also less stable than wild-type Mec1 and demonstrated reduced nuclear localization. We also identified rpn3-L140P, which encodes a component of the 19S proteasomal regulatory particle of the 26S proteasome, as a suppressor of the temperature-sensitive growth caused by mec1-W2368A.
As Cdt1 is required for the nuclear import and origin loading of Mcm2-7, we also sought to investigate the interaction between these two components in more detail. Using reconstituted Mcm2-7·Cdt1 complexes from bacterial-expressed proteins, we demonstrated that these complexes exhibit lower ATPase and helicase activity than Mcm2-7. We also showed that Mcm2-7 dissociates into subcomplexes, and that Mcm3, 5 and 7 bound origins in the absence of Cdt1. We propose that the reduced ATPase activity of Mcm2-7 by Cdt1 binding is induced by structural changes in the Mcm2-7 ring. We also suggest that Cdt1 helps to stabilize the Mcm2-7 hexamer.
To investigate the role of phosphorylation on Mcm2-7, we utilized a phosphomimetic mutant of Mcm4 that when incorporated into Mcm2-7 can bypass the requirement for DDK. While phosphomimetic Mcm4 demonstrated slightly lower ATPase activity than the wildtype protein, phosphomimetic Mcm2-7 complexes exhibited wildtype ATPase, helicase and DNA binding activity.
Taken together, our work identifies the functional role of the C-terminal residues of Mec1 and the protein’s turnover by the proteosome. Our studies also provide new insights into the factors and processes involved in the activation of Mcm2-7 to unwind DNA.
DaSilva, Lance F., "Regulation of eukaryotic Mcm2-7 activity" (2013). Electronic Thesis and Dissertation Repository. 1766.