Practice question

Eukaryotic replication origins must fire exactly once per cell cycle, a requirement enforced by strict licensing control. In G1, origin recognition complex recruits Cdc6 and Cdt1 to load MCM2-7 double hexamer helicases to form pre-replicative complexes. Upon S-phase entry, CDK and DDK activations fire origins and simultaneously trigger elimination of licensing factors to prevent re-assembly. Geminin provides a crucial additional layer independent of phosphorylation. Synthesized from S phase onward and stable through G2 and early mitosis, geminin directly binds coiled-coil domain of Cdt1 with high affinity, masking the MCM-interacting surface and blocking recruitment of additional MCM complexes to already replicated origins. Accumulation is cell cycle regulated: in late mitosis and G1, APC/C-Cdh1 polyubiquitinates geminin for proteasomal destruction, releasing Cdt1 for new licensing only when low CDK environment is appropriate. Depletion of geminin induces rereplication leading to double-strand breaks, checkpoint activation and apoptosis, while overexpression blocks S phase, demonstrating essential role as inhibitor of origin re-licensing ensuring once-per-cycle duplication. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.