Soon after Cdt1 overexpression, RPA-bound ssDNA is accumulated, activating ATR prior to the detection of extensive re-replication, whereas ATM activation occurs when DSBs are detected at much later stages. This observation is consistent with the hypothesis that ATR is activated when the initial steps of DNA re-replication are detected, and this activation in turns suppresses further DNA re-replication. Given the critical roles of the S-phase checkpoint in the suppression of DNA re-replication, what are the signals triggering the activation of the S-phase checkpoint upon loss of replication licensing? When Cdt1 is overexpressed or Geminin expression is lost, Cdt1 re-loads MCM proteins onto replication forks and reassembles pre-RCs in S-phase (Figure 3). Inhibition of re-replication initiation by overexpressing p27 or by inhibiting Cdc7 kinase activity prevents S-phase checkpoint activation