All organisms ensure once and only once replication during S phase through a 
process called replication licensing. Cdt1 is a key component and crucial 
loading factor of Mcm complex, which is a central component for the eukaryotic 
replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin 
is essential to prevent rereplication. Here, we address the mechanism of DNA 
licensing using purified Cdt1, Mcm and Geminin proteins in combination with 
replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse 
Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 
346th arginines constituting an arginine finger-like structure to alanine 
(RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication 
activity in Cdt1-depleted extracts but also its specific activity for DNA 
replication and licensing was significantly increased compared to the wild-type 
protein. In contrast, the R223 mutants were partially defective in rescue of DNA 
replication and licensing. Biochemical analyses of these mutant Cdt1 proteins 
indicated that the RR-AA mutation disabled its functional interaction with 
Geminin, while R223 mutations resulted in ablation in interaction with the 
Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the 
phosphorylation-induced inactivation or chromatin dissociation. Our results show 
that conserved arginine residues play critical roles in interaction with Geminin 
and Mcm that are crucial for proper conformation of the complexes and its 
licensing activity.