UC San Diego

S-phase DNA replication critically maintains stability of the eukaryotic genome, and is regulated at its initiation phase by pre-replicative (pre-RC) complex formation in G1 and activation at G1/S. Pre-RC component, Cdc6, was previously presumed to be dispensable after S-phase entry. Recent studies show a continued presence of Cdc6 during S- phase and later in the mammalian cell cycle, implicating further cell cycle function. In the first section of III. RESULTS, I investigated the Cdc6 function in S-G2/M. Small interference RNA-mediated depletion of Cdc6 in asynchronous HeLa cells induces G1/S arrest and elevated cell death. Cdc6 depletion in G1-synchronized HeLa cells blocks S-phase entry, confirming its critical pre-RC assembly function in G1. However, Cdc6 depletion in synchronous S-phase HeLa cells slows DNA replication and leads to mitotic lethality. S-phase Cdc6 depletion reduces new origin firing, whereas progressing forks remain active. This abnormal replication does not activate conventional S-phase replication checkpoint response, and ultimately, cells enter mitosis with abnormally condensed chromosomes and spindle structures, and undergo mitotic cell death. Thus, not only is Cdc6 essential for G1 origin licensing, it is also required in S-phase for proper origin firing and replication dynamics that are essential for viable cell cycle progression. Intriguingly, pre-RC deficiency induces cell death in cancer cells, but not in untransformed mammalian cells. In the third part of III. Results, I investigated the role of pre-RCs in checkpoint response, and the basis for this phenotypic paradigm during pre-RC deficiency. During pre-RC deficiency, untransformed and cancer cells arrest in G1, due to abrogated pre-RC assembly/replication initiation. However, pre-RC deficiency in S-phase activates a crucial ATR- dependent checkpoint response in untransformed cells that specifically suppresses progressing fork activity and induces cell cycle arrest, but not in cancer cells, which permit abnormal replication that alters chromatin structure and results in cell death. The lack of cancer cell response to pre-RC deficiency is attributed to elevated tolerance for abnormal replication structures. Lack of response to pre-RC-deficiency in cancer cells represents an altered relationship between pre-RC and checkpoint proteins and DNA replication, and holds significant anti-cancer therapeutical potential