Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21WAF1, a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21WAF1 in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21+/+ cells stably arrest in G2, whereas p21−/− cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21+/+ cells. Inactivation of APC in irradiated p21+/+ cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21−/− cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage
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