DETERMINING THE CELLULAR AND MOLECULAR CONSEQUENCES OF NATURALLY-OCCURRING AND ENGINEERED MUTATIONS IN THE CELL CYCLE GENE CDT1

The cell division cycle is composed of two major events; 1. DNA replication (S phase) and 2. chromosome segregation (M phase). In addition, gap phases exist to prepare cells for the duplication of their entire genome (G1 phase) and to check that the genome has been faithfully duplicated before cell division (G2 phase). This entire process involves many steps each governed by a set of molecular mechanisms that allow cells to transition from one cell cycle phase to the next. How these mechanisms are arranged and linked to intra- and extracellular cues for timely and coordinated cell division is a topic of great interest in the field. In this work, we focus on a critical step for cell cycle progression, known as origin licensing, with emphasis on elucidating the molecular function of Cdt1. The Cdt1 protein is highly conserved in metazoans and its role is essential for origin licensing, yet the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than WT Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex position the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both Cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live cell imaging analysis demonstrated no change in stability of this variant however. Instead, we propose that Cyclin A/CDK inhibits Cdt1 licensing function independently of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability. In addition, we present here recent advances in elucidating Cdt1 molecular functions in origin licensing as well as describe the current understanding of human Cdt1 regulation.Doctor of Philosoph