The correct execution of DNA replication is critical for normal cell proliferation. The earliest step in DNA replication is known as origin licensing. Origins are licensed by the loading of MCM complexes, an event that is restricted to G1 phase. The Cdt1 protein is essential for MCM loading, yet the molecular mechanisms of its activity are still remarkably mysterious. Here, we examined a collection of rare Cdt1 variants associated with a specific form of human primordial dwarfism (Meier‐Gorlin syndrome), plus one hypomorphic conserved Drosophila allele, to shed light on Cdt1 function. Our results indicate that two dwarfism variants (Cdt1‐R462Q, Cdt1‐E468K) are expressed normally, but load less MCM than WT Cdt1. We found the lower origin licensing activity of these variants correlates with impaired MCM binding. Likewise, the hypomorphic Drosophila allele (Cdt1‐R210C) consistently induces less MCM loading, and this deficiency also correlates with impaired MCM binding. Interestingly, this variant impacts a previously uncharacterized MCM binding domain in Cdt1. To visualize these molecular interactions, we generated human Cdt1‐MCM structural homology models based on recent yeast cryo‐EM structures. Our models position the hypomorphic mutations at direct contact points in the interface between human Cdt1 and human MCM complex. These results suggest human Cdt1 uses two domains to make multiple contacts with the MCM hexamer rather than the previously‐described single Cdt1‐MCM interaction site. Furthermore, this newly‐discovered MCM binding domain can explain how the Cdt1 inhibitor protein, geminin, blocks Cdt1‐MCM interaction.Surprisingly, one dwarfism allele (Cdt1‐A66T) is more rather than less active than WT Cdt1, reproducibly inducing four‐fold more DNA re‐replication when overexpressed to the same degree. A66 is located next to the S phase CDK binding motif, and its mutational alteration inhibits Cyclin A binding and subsequent recognition by the E3 ubiquitin ligase, SCFSkp2. This variant is not more stable however and has identical degradation and accumulation kinetics to WT Cdt1, presumably because the CRL4Cdt2 E3 ubiquitin ligase interaction is intact. We postulate that Cyclin A acts as a direct inhibitor of Cdt1‐MCM binding, suggesting a novel Cyclin‐dependent mechanism of Cdt1 inhibition at the end of S phase and prior to cell division. Together, these findings reveal additional roles of Cyclin/Cdk regulation of Cdt1, as well as newly described Cdt1‐MCM molecular interactions. Overall, our study identifies two new aspects of Cdt1 interactions that ensure efficient but once‐and‐only once genome duplication and normal cell proliferation.Support or Funding InformationThis work was supported by the National Institutes of Health award to J.G.C. R01GM102413 and R01GM102413‐S1 and to the University of North Carolina Flow Cytometry Core Facility award P30CA016086. P.N.P. is supported by the National Institutes of Health F31GM121073.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.