Studies on the Initiation Stage

We have cloned ORC from multiple sources, including S. pombe (Sp), human (h) and S. cerevisiae (Sc). The properties of these complexes differ as do their interactions with DNA. The SpORC and ScORC contain stoichiometric levels of six distinct subunits while hORC contains near stoichiometric levels of ORC 1-5 subunits but only low levels of the ORC6 subunit. Both h and ScORC, require ATP for DNA binding whereas SpORC does not. SpORC contains a modified 140 kDa ORC4 subunit with a unique N-terminal region that includes multiple repeats of “AT-hooks” that bind strongly to AT-rich regions. The binding of SpORC to DNA occurs solely through this N-terminal ORC4 domain. Direct visualization of SpORC binding to origin DNA using atomic force microscopy demonstrated that the DNA is wrapped around the ORC4 subunit. DNA wrapping around the ORC4 subunit was detected with both the six subunit ORC as well as with the ORC4 subunit alone. Treatment of the SpORC (or ORC4 subunit)-origin DNA complex with topoisomerase I induced a negative shift in the topoisomer distribution. Thus binding of SpORC origin to DNA alters the structure of DNA. Similar findings have been observed qualitatively with h and Sc ORC. The ORC-induced alteration of DNA, however, does not result in melting of the duplex, an event that is required for subsequent duplex unwinding and replication reaction.

Efforts are underway to examine whether the formation of the Pre-RC leads to the melting of the DNA duplex For this purpose, we have cloned and isolated the Cdc6p (SpCdc18p), Cdt1p and the Mcm six-subunit complex. Direct interactions of purified Cdc6p and Cdt1p with origin DNA depends on the presence of ORC while the addition of the Mcm complex requires the addition of crude extracts derived from cells lacking the cyclin-dependent protein kinase activity (G1 extracts). Attempts to reconstitute formation of the Pre-RC with purified proteins is now underway.

The Pre-RC, containing ORC, Cdc6p, Cdt1p and the Mcm six-subunit complex is then converted to the pre-initiation complex (Pre-IC) by a series of interactions that require its association with Mcm10p, GINS (a four subunit complex), Sld3p and Cdc45p. In addition, this step occurs coincidental with the activation of at least two protein kinases, CDKs and Cdc7p-Dbf4p. During this remodeling step, both Cdt1p and Cdc6p are modified by phosphorylation and removed from the origin complex . Critically, the loading of Cdc45p leads to the activation of the unwinding of duplex DNA carried out by the Mcm complex.

We have cloned a number of the proteins involved in formation of the Pre-IC complex and their characterization is presently under investigation. The activation of the helicase activity associated with the Mcm2-7 complex is of prime interest. In all eukaryotes, the Mcm complex contains six distinct proteins, Mcm 2 to 7. In vivo studies demonstrated that this complex is associated with origins at G1 (with ORC) but during replication, the Mcm 2-7 complex is associated with the replication fork. Inactivation of any one of the Mcm proteins blocks continued fork progression. The cloned Mcm2-7 complex, isolated from all eukaryotes, is devoid of ATPase or DNA helicase activity while a subcomplex containing the Mcm4, 6 and 7 subunits contains both activities. The addition of the other Mcm proteins (Mcm2, 3 and 5) to the Mcm 4, 6 and 7 complex, leads to the formation of the Mcm2-7 complex and results in the loss of ATPase and DNA helicase activity. The role of Cdc45p and protein kinases essential for the activation of the cryptic helicase activity residing in the Mcm2-7 complex is now being investigated. We have demonstrated that the Mcm2-7 complex, a known target of the Cdc7p-Dbf4p protein kinase, interacts with Mcm10p. Our laboratory has shown that Mcm10p participates in a number of key interactions including those required for both the initiation and elongation stages of replication. These include its interaction with the Cdc7p-Dbf4p complex which markedly stimulates the phosphorylation of the Mcm2-7 complex, its role in loading of Cdc45p to form the Pre-IC and its direct interaction with the catalytic subunit of DNA polymerase a which markedly stimulates its polymerase activity. The role of the multifunctional Mcm10p in the elongation phase of replication is also under investigation.