Despite the differences in the DNA sequences that determine the position of replication origins in yeast and higher eukaryotes, the proteins determining the initiation site are well conserved. Proteins homologous to the subunits of ORC have
been identified in a number of higher eukaryotes. First identified were Drosophila
Orc2p and Orc5p proteins (Gossen et al., 1995). The genes encoding these proteins
were initially identified by analysis of the Drosophila genome project. The sequence
conservation between the Drosophila and S. cerevisiae ORC protein is relatively low,
with 21% identity between the Orc2p proteins from the two species and Drosophila
Orc5p has 22% identity with the S. cerevisiae Orc5p (Gossen et al., 1995). It was the
low degree o f conservation between ORC subunits from different species, which made their identification in higher eukaryotes difficult.
Drosophila Orc2p and Orc5p proteins were found to be complexed with four other
proteins, suggesting the presence of an Origin Recognition Complex similar to that
from yeast (Chesnokov et a l , 1999; Gossen et a l , 1995). Microsequencing of three
co-purified proteins led to the identification of Drosophila Orc4p which is 24%
identical to S. cerevisiae Orc4p, Orc3p with 21% identity to the yeast counterpart and
Orc6p which is 19% identical to the S. cerevisiae Orc6p (Chesnokov et aL, 1999).
The Drosophila homologue of Ore Ip was also found to be part o f the Drosophila
ORC (Chesnokov et a l , 1999; Pak et a l , 1997). Interestingly, Drosophila Orc2 was
independently identified as a gene whose mutation reduced chorion amplification in ovarian follicle cell, implying a function for ORC in the initiation of DNA replication in Drosophila (Landis et a l , 1997).
A fragment o f human Ore Ip was identified in two-hybrid screen for proteins interacting with the herpes simplex virus yICP34.5 protein and consequently used to
isolate full length Xenopus Ore Ip (Rowles et al., 1996). Xenopus Orc2p was
identified in a screen for inhibitors of cdc2 kinase activity that prevented mitotic
catastrophe (Carpenter et al., 1996). The purification and microsequencing of
proteins associated with Xenopus Ore Ip and Orc2p led to the identification of
Xenopus Orc3p and Orc4p (Carpenter and Dunphy, 1998; Tugal et a l , 1998).
Aminoacid identities between S. cerevisiae and Xenopus Ore Ip, Orc2p, Orc3p and
Orc4p are 20%, 23%, 14% and 22% respectively (Carpenter and Dunphy, 1998; Carpenter et a l , 1996; Rowles et a l , 1996; Tugal et a l , 1998).
Human Ore Ip and Orc2p were identified by degenerate PCR (Gavin et a l , 1995).
Human Ore Ip is 27% identical to the S. cerevisiae protein. Human Orc4p was
identified by searching EST databases and was found to be 29% identical to the Orc4p from S. cerevisiae (Quintana et a l , 1997; Tugal et a l , 1998). To identifiy
C hapter 1
human Orc5p, databases were searched with peptide sequences derived from a
polypeptide co-purifying with Xenopus Ore Ip (Tugal et al., 1998). These searches
identified human Orc5p which has 29% identity to the yeast version o f the gene (Ishiai etal., 1997; Quintana etal., 1998; Tugal etal., 1998).
The identification of ORC subunit homologues in several higher eukaryotes suggests the presence o f a complex with a function in origin recognition similar to that of
S. cerevisiae ORC. ORC subunits in Drosophila and Xenopus are found in high
molecular weight complexes (Carpenter and Dunphy, 1998; Carpenter et a l , 1996;
Chesnokov et al., 1999; Gossen et a l , 1995; Romanowski et a l , 1996b; Rowles et
a l , 1996; Tugal et a l , 1998). Individual ORC subunits were also found to co-
imunoprecipitate from human extracts (Quintana et a l , 1997; Quintana et a l , 1998).
Moreover, purified ORC complexes from Xenopus and Drosophila were able to
restore DNA replication in ORC depleted Xenopus extracts establishing the crucial
role of ORC in DNA replication in higher eukaryotes (Chesnokov et a l , 1999;
Romanowski et a l , 1996b; Rowles et a l , 1996). A function for ORC in DNA
replication in higher eukaryotes is also supported by the fact that certain Orc2p
mutants of Drosophila are defective in chorion locus amplification (Landis et a l ,
1997). Interestingly, one copy of human Orc5 gene has been found to be deleted in uterine leiomyomas, which are benign tumours that rarely progress to malignancy
(Quintana et a l , 1998). It is possible that deletion of one copy of Orc5 slows the cell
cycle, preventing abundant proliferation and genomic instability leading to malignancy.
The identification o f ORC complexes from higher eukaryotes provided a new prospective on the process of origin specification. Despite no clear DNA sequence serving as an origin of DNA replication being identified in higher eukaryotes.
complexes similar to those in S. cerevisiae are clearly required for DNA replication.
This allowed studying origin specification in higher eukaryotes by investigating the
sequence elements recognised by ORC. In D ro so p h ila , in vivo and in vitro
approaches have been used to demonstrate that Drosophila ORC binds to replication
elements that direct initiation in the chorion gene cluster (Figure 1/6, Austin et a l ,
1999). The ACE element, previously identified to be required for the ability of the DNA fragment from the chorion gene cluster to confer initiation competence to novel chromosomal location, was shown to direct immunolocalisation of ORC in follicle
cells. In vivo cross-linking and chromatin immunoprécipitation also demonstrated
association o f ORC with ACE and with AER-d (Figure 1/6). Moreover, purified
Drosophila ORC binds to ACE and AER-d in vitro, and like its S. cerevisiae
counterpart, this binding is dependent on ATP. These experiments for the first time demonstrated that ORC from higher eukaryotes binds specifically to the DNA sequence which serve as an origin of DNA replication. In the view of these results, it
is likely that ORC in Xenopus somatic cells recognises DNA sequence elements
within the replication origins of Xenopus. The random initiation of DNA replication
in Xenopus egg extracts remains however unexplained. The possible explanations for
this apparent paradox include embryonic ORC being different to the somatic ORC or ORC levels in the egg being extremely high, driving ORC towards non-specific ORC-DNA interactions.