Replication origin licensing deficiency and genome instability during cell cycle re‐entry from quiescence

Abstract

Metazoan cells transition between active cell division cycles and periods of quiescence during development, regeneration, and homeostasis. Prior to cell division, DNA replication initiates during S phase at thousands of origins to ensure timely and complete genome duplication. Each origin that initiates must have been already licensed for replication in the preceding G1 phase by the DNA loading of Minichromosome Maintenance (MCM) complexes. Insufficient origin licensing in G1 causes incomplete replication and is associated with hypersensitivity to replication stress and DNA damage. We hypothesize that physiological differences in G1 length or G1 starting point impact origin licensing dynamics, and that such differences affect genome stability. We have explored origin licensing parameters in the first G1 phase during cell cycle re‐entry from quiescence relative to the G1 phases following mitosis in actively proliferating cells. We used single cell flow cytometry and live cell imaging to define the relationships among cell cycle re‐entry, G1 length, origin licensing dynamics and replication stress in untransformed human epithelial cells. Despite spending a long time in G1 phase, cells re‐entering the cell cycle are significantly underlicensed in the first cell cycle but not the second or subsequent cell cycles. Cells enter their first S phase before licensing is complete and are then hypersensitive to replication stress. Thus, G1 phase upon cell cycle re‐entry is characterized by slow (or delayed) origin licensing and an apparently weak licensing checkpoint. Strikingly, repeated transitions into and out of quiescence and active proliferation exacerbate replication stress and cause hypersensitivity to replication inhibitors Therefore, the transition from quiescence to active division is a particularly sensitive time for genome stability, and the number of transitions into and out of quiescence may play a role in long term cell viability and genome stability.Support or Funding InformationThe UNC Flow Cytometry Core Facility is supported in part by P30 CA016086. This work was supported by a fellowship from the NSF (DGE‐1144081) to J.P.M and by grants from the NIH to J.G.C. (GM083024 and GM102413).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.