Interaction in vitro; protein produced in a baculovirus and insect cell system.

Crystal structure of hexameric complex captured as 15 pairwise interactions.

Interaction in vitro; protein produced in a baculovirus and insect cell system.

Binding of Orc6 to preassembled Orc1-5 complex captured as 5 pairwise interactions.

Interaction in vitro; bait protein produced in a baculovirus and insect cell system.

A236C mutant of Orc6 used. Based on crystal structure, a Cys at residue 236 of Orc6 would be predicted to crosslink with Cys 372 of Orc3.

Embryonic nuclear extract was treated with DNase and RNase.

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Embryonic nuclear extract was treated with DNase and RNase.

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Only Orc3 and Orc6 were co-infected (no other Drosophila ORC subunits included).

Source was cell extract of baculovirus-infected High5 cell line; bait produced from transfected construct; prey produced from transfected construct.

The Orc3-Orc6 interaction is probably direct. In a similar experiment, no interaction was observed between Orc2 and Orc6. The Y225S mutation corresponds to the Meier-Gorlin-Syndrome mutation.

His-Orc1, Orc2, Orc3, Orc4, Orc5 and MBP-Orc6 were co-infected, then purified by sequential His-Orc1 then MBP-Orc6 pull down with additional preparative gel filtration purification.

The Drosophila origin of replication complex (ORC) forms a stable, hexameric complex containing subunits Orc1, Orc2, Orc3, Orc4, Orc5 and Orc6. Binding of Orc6 to Orc3 is probably direct as it is observed when only these two ORC proteins are co-infected.

Source was cell extract of baculovirus-infected High5 cell line; bait produced from transfected construct; prey produced from transfected construct.

The ORC complex visualized by EM was captured as 15 pairwise interactions. The addition of ATP caused the complex to adopt an active/open conformation.

Interaction in vitro; proteins produced as a recombinant fusion proteins in baculovirus and High5 cell system.

Comparison of EM structures of ORC complex, ORC complex bound to DNA and a ternary complex consisting of ORC, DNA and Cdc6 curated as a series on binary protein-protein interactions.

Interaction in vitro; proteins produced as recombinant fusion proteins in a baculovirus and insect cell system.