Accurate DNA replication requires a complex interplay of many regulatory 
proteins at replication origins. The CMG (Cdc45·Mcm2-7·GINS) complex, which is 
composed of Cdc45, Mcm2-7, and the GINS (Go-Ichi-Ni-San) complex consisting of 
Sld5 and Psf1 to Psf3, is recruited by Cdc6 and Cdt1 onto origins bound by the 
heterohexameric origin recognition complex (ORC) and functions as a replicative 
helicase. Trypanosoma brucei, an early branched microbial eukaryote, appears to 
express an archaea-like ORC consisting of a single Orc1/Cdc6-like protein. 
However, unlike archaea, trypanosomes possess components of the eukaryote-like 
CMG complex, but whether they form an active helicase complex, associate with 
the ORC, and regulate DNA replication remains unknown. Here, we demonstrated 
that the CMG complex is formed in vivo in trypanosomes and that Mcm2-7 helicase 
activity is activated by the association with Cdc45 and the GINS complex in 
vitro. Mcm2-7 and GINS proteins are confined to the nucleus throughout the cell 
cycle, whereas Cdc45 is exported out of the nucleus after DNA replication, 
indicating that nuclear exclusion of Cdc45 constitutes one mechanism for 
preventing DNA re-replication in trypanosomes. With the exception of Mcm4, Mcm6, 
and Psf1, knockdown of individual CMG genes inhibits DNA replication and cell 
proliferation. Finally, we identified a novel Orc1-like protein, Orc1b, as an 
additional component of the ORC and showed that both Orc1b and Orc1/Cdc6 
associate with Mcm2-7 via interactions with Mcm3. All together, we identified 
the Cdc45·Mcm2-7·GINS complex as the replicative helicase that interacts with 
two Orc1-like proteins in the unusual origin recognition complex in 
trypanosomes.