Delayed activation of the DNA replication licensing system in Lgr5(+) intestinal stem cells

During late mitosis and early G1, replication origins are licensed for replication by binding to MCM2-7 double hexamers. This signals proliferative fate commitment. Here, we investigate how licensing and proliferative commitment are coupled in the small-intestinal epithelium. We developed a method for identifying cells in intestinal crypts that contain DNA-bound MCM2-7 and are licensed for replication. Interphase cells at the top of the transit amplifying zone did not contain DNA-bound MCM2-7, but still expressed MCM2-7 protein. This suggests licensing is inhibited immediately at terminal differentiation, after a final mitosis. Strikingly, we found that at the crypt base the majority of Lgr5(+) intestinal stem cells reside in an unlicensed state, despite expressing MCM2-7 protein and the Ki67 proliferation marker. This state, which we call shallow-G0, might allow stem cells to be easily activated to re-enter the cell cycle. We demonstrate that the dynamics of the licensing system provides a novel means to assess the unique cell-cycle of intestinal epithelial cells.