Initiation of DNA replication depends upon recog-
nition of genomic sites, termed origins, by AAA+
ATPases. In prokaryotes a single factor binds each
origin, whereas in eukaryotes this role is played
by a six-protein origin recognition complex (ORC).
Why eukaryotes evolved a multisubunit initiator, and
the roles of each component, remains unclear. In
Trypanosoma brucei
, an ancient unicellular eukary-
ote, only one ORC-related initiator, TbORC1
/
CDC6,
has been identified by sequence homology. Here
we show that three TbORC1
/
CDC6-interacting fac-
tors also act in
T. brucei
nuclear DNA replication
and demonstrate that TbORC1
/
CDC6 interacts in a
high molecular complex in which a diverged Orc4
homologue and one replicative helicase subunit can
also be found. Analysing the subcellular localization
of four TbORC1
/
CDC6-interacting factors during the
cell cycle reveals that one factor, TbORC1B, is not
a static constituent of ORC but displays S-phase
restricted nuclear localization and expression, sug-
gesting it positively regulates replication. This work
shows that ORC architecture and regulation are di-
verged features of DNA replication initiation in
T. bru-
cei
, providing new insight into this key stage of eu-
karyotic genome copying.