Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Megan Davey

Abstract

The transmission of genetic information from parental to daughter cells requires the faithful duplication of an organism’s genome. Uncontrolled DNA replication can result in proliferative diseases, such as cancer. DNA replication requires a single-stranded DNA template to be produced from duplex DNA. In eukaryotes, DNA unwinding for replication is performed by the heterohexameric replicative helicase complex comprised of the minichromosome maintenance proteins 2 through 7.

Each of the Mcm2-7 subunits likely has a unique role in DNA binding and unwinding by the Mcm2-7 complex. The present study examines the role of the Saccharomyces cerevisiae Mcm2 subunit in regulating the activities of Mcm complexes. Using in vitro assays for DNA unwinding and DNA binding with E. coli-purified Mcm subunits, this work demonstrates that Mcm2 requires nucleotide to actively regulate DNA binding and unwinding by Mcm complexes. These studies define Mcm2 as an active regulatory subunit within the Mcm2-7 complex.

Mcm2-7 is also targeted by various kinases that presumably modulate its activities. The Dbf4-dependent kinase (DDK) comprised of the Cdc7 catalytic subunit and Dbf4 regulatory subunit is one such kinase. Here, the residues of Mcm2 targeted for phosphorylation by DDK were mapped using recombinant proteins and verified in cells. The effects of phosphomimetic substitutions at these positions on the activities of the Mcm2-7 complex were examined. Interestingly, the ATPase activity of Mcm2 of the phosphomimetic Mcm2 is lower compared to wild type. A version of Mcm2-7 containing the phopshomimetic mutant of Mcm2 had a higher affinity for DNA, which in turn inhibited DNA unwinding by the complex. The biological function of phosphorylation of Mcm2 by DDK in budding yeast was also examined using cells containing a version of mcm2 that cannot be phosphorylated by DDK. This mutation rendered the cells sensitive to agents that cause DNA base damage. Additionally, the mutant allele interacted with genes involved in the DNA damage checkpoint as determined by synthetic genetic array analysis. In sum, a model in which DDK-dependent phosphorylation of Mcm2 regulates its ATPase activity to slow replication forks in the cell’s response to DNA damage is proposed.


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Biochemistry Commons

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