Recently, several novel members have joined the family of pre-RC (Figure
) and pre-IC (Figure
), mostly discovered from proteomic screens/analyses and bioinformatic predictions; at the same time, many classic factors, including HBO1, 14-3-3, HOX, MCM10, FACT, Ctf4, and Y RNAs, were merited with additional roles in replication.
Figure 1 Emerging players in the assembly of pre-replicative complex (pre-RC). The classical model of pre-RC assembly involves the ordered loading of ORC, Cdc6, Cdt1 and MCM2-7 onto replication origins. The Cdt1 inhibitor, Geminin associates with Cdt1 outside (more ...)
Figure 2 Emerging players in the assembly of pre-initiation complex (pre-IC) and replisome progression complex (RPC). Other than classical factors in pre-IC and RPC (Cdc45, MCM2-7, GINS, RecQ4, TopBP1, and DNA polymerase), a number of novel factors have recently (more ...)
It is notable that different players exhibit diverse conservation levels across the organisms. For some proteins like Ctf4, their functions are faithfully reproduced in all model organisms investigated. For some novel players, such as ORCA/LRWD1, whether orthologs exist or have conserved functions in other eukaryotic species is still an open question. For other factors like MCM9, disparate observations have been made in different systems, which could either reflect technical difference, or indicate real evolutionary divergence.
It is also noteworthy that many of these factors are involved in other non-replication events, evident from ORCA/LRWD1 in heterochromatin silencing [20
], 14-3-3 in various signaling pathways [40
], HOX in developmental control [53
], FACT in transcription elongation [145
], Ctf4 in sister chromatid cohesion [159
], and Treslin/Ticrr in G2/M checkpoint [130
]. It is possible that these factors may have replication-independent functions, or may couple replication to additional processes to form a regulatory network.
What we know is probably only the tip of the iceberg. For instance, the complete in vitro
reconstitution of DNA replication initiation machinery or sub-complexes using purified proteins has so far been unsuccessful, indicating that there are still important players missing from the orchestra. Investigations in other less-studied species may lead us to some valuable clues. In Tetrahymena thermophila
, ORC has a 26T RNA component that is involved in ribosomal DNA origin recognition via complementary base pairing [193
]. It will be interesting to find out whether a similar functional player exists in other model organisms. On the other hand, with the increasing genome size and complexity of protein network from lower to higher eukaryotes, how timely control and dynamic regulation of replication are achieved also need further elucidation. As reported from a recent study using a novel hybrid CHO cell/Xenopus
egg extracts, a pre-restriction point inhibitor exists for licensing. Other than ruling out pre-RC components, Geminin, HBO1 or CDK, the nature of this inhibitor was not characterized [194
]. Therefore, exploring new model systems and utilizing innovative techniques, along with mechanistic studies on existing factors, will help us probe for the missing links and mechanisms, toward a better grasp of the final truth on how eukaryotes initiate DNA replication.