Function of the origin recognition complex 1 (ORC1) outside DNA replication in Drosophila

Abstract

The origin recognition complex (ORC) is an essential component of the pre-replicative complex (pre-RC) that binds to replication origins for licensing. Levels of the largest ORC subunit, ORC1, oscillate during the mitotic cell cycle and regulate origin usage. In Drosophila, ORC1 levels increase at the G1/S transition following E2F-dependent transcriptional activation, remain high until the end of M phase and then decrease at the M/G1 transition when ORC1 is targeted for proteolysis by the anaphase-promoting complex (APC). A function, if any, for Drosophila ORC1 after S phase has not been described. Here, we determined the role of ORC1 at stages outside S phase by generating ORC1 derivatives with a modified ORC1 degradation box (the O-box) and examining the effects in vivo. These modifications either stabilized ORC1 by mutating the O-box (ORC1Omut) so that it is no longer targeted by APC or changed its degradation profile by replacing the O-box with the D-box of human cyclin B (ORC1O→D), so that degradation would occur earlier. We determined the distribution and tested the function of these ORC1 derivatives in an orc1 mutant background so that only the mutated protein was expressed. Stable version of ORC1, ORC1Omut, showed no effects on cell cycle progression; however, ORC1O→D, which is degraded early at the G2/M transition, led to a higher frequency of M-phase cells but not S-phase cells. Taken together, our results indicate the timing of ORC1 degradation is required for timely progression in M phase.