Matson et al. find that human cells re-entering the cell cycle from quiescence have an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress.

The authors find that human cells re-entering the cell cycle from quiescence have both an impaired p53-dependent DNA replication origin licensing checkpoint and slow origin licensing. This combination makes every first S phase underlicensed and hypersensitive to replication stress. ABSTRACTTo maintain tissue homeostasis, cells transition between cell cycle quiescence and proliferation. An essential G1 process is Minichromosome Maintenance complex (MCM) loading at DNA replication origins to prepare for S phase, known as origin licensing. A p53-dependent origin licensing checkpoint normally ensures sufficient MCM loading prior to S phase entry. We used quantitative flow cytometry and live cell imaging to compare MCM loading during the long first G1 upon cell cycle entry and the shorter G1 phases in the second and subsequent cycles. We discovered that despite the longer G1 phase, the first G1 after cell cycle re-entry is significantly underlicensed. As a result, the first S phase cells are hypersensitive to replication stress. This underlicensing is from a combination of slow MCM loading with a severely compromised origin licensing checkpoint. The hypersensitivity to replication stress increases over repeated rounds of quiescence. Thus, underlicensing after cell cycle re-entry from quiescence distinguishes a higher risk cell cycle that promotes genome instability.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E–associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.

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.

Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Cyclin E overproduction is toxic to mammalian cells, although the gene encoding cyclin E (CCNE1) is overexpressed in some cancers. It is not yet understood how cancer cells tolerate high levels of cyclin E. To address this question, we extensively characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing human cyclin E briefly experienced truncated G1 phases, then consistently endured a transient period of DNA replication origin underlicensing, replication stress, and severely impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in both cell cycle dynamics and CDK activity. Each of these phenotypes improved rapidly despite maintaining high cyclin E-associated activity. Transcriptome analysis revealed that adapted cells downregulated a cohort of G1-regulated genes. These cells also shared at least one unique change also found in breast tumors that overproduce cyclin E, expression of the cancer/testis antigen HORMAD1. Withdrawing cyclin E induction partially reversed the intermediate licensing phenotype of adapted cells indicating that adaptation is at least partly independent of genetic alterations. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing by an indirect mechanism through premature S phase onset. It serves as an example of specific oncogene adaptation that can identify key molecular changes during tumorigenesis.