Data for: Cdt1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis

Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor Cdt1 after origin firing, raising the question of how cells prevent re-replication before Cdt1 is fully degraded. Here, using quantitative microscopy and in vitro reconstituted human DNA replication, we show that Cdt1 inhibits DNA synthesis during an overlap period when Cdt1 is still present after origin firing. Cdt1 inhibits DNA synthesis by suppressing CMG helicase progression at replication forks, and DNA synthesis commences once Cdt1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.