SARC Award Slide Deck

Origin licensing occurs during G1, when inactive minichromosomal maintenance (MCM) complexes are loaded onto chromatin to prepare cells for DNA replication. Human cells normally license more origins than needed, creating dormant origins that can rescue replication when forks slow or stall. Because licensing is restricted to G1, lengthening G1 may increase MCM loading and generate hyperlicensed cells with a larger dormant origin pool. This project investigates whether hyperlicensing alters the response of OVCAR-3 ovarian carcinoma cells to replication stress during S phase. The OVCAR-3 cells used have enhanced Cyclin E activity through CCNE1 amplification and high p16 expression, making them dependent on CDK2 for G1/S progression. We use the CDK2 inhibitor INX-315 to hold cells in G1 and promote hyperlicensing, then release them into S phase. Replication stress is induced with hydroxyurea, a ribonucleotide reductase inhibitor, and measured using γH2AX, a marker of replication-associated DNA damage. By optimizing hydroxyurea dose and timing, we aim to determine whether hyperlicensed cells are more resistant or hypersensitive to replication stress.